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Sample records for keratinocyte growth factor

  1. Acidic fibroblast growth factor and keratinocyte growth factor stimulate fetal rat pulmonary epithelial growth.

    PubMed

    Deterding, R R; Jacoby, C R; Shannon, J M

    1996-10-01

    We have shown that pulmonary epithelial growth and differentiation can occur if pulmonary mesenchyme is replaced with a mixture of growth factors [total growth medium (TGM)] that consists of adult rat bronchoalveolar lavage fluid, insulin, epidermal growth factor (EGF), cholera toxin (CT), acidic fibroblast growth factor (aFGF), and fetal bovine serum. In the present study, we have defined the importance of specific components of TGM. Day 14 fetal rat distal lung epithelium, devoid of mesenchyme, was enrobed in growth factor-depleted Matrigel and cultured for 5 days in various soluble factors. We found that deleting aFGF or CT from TGM significantly reduced DNA synthesis. Epithelial proliferation was not significantly different when keratinocyte growth factor (KGF) replaced aFGF in TGM. KGF, however, required the presence of a basal medium containing CT, insulin, and serum for optimal proliferation. We then added specific growth factors to the basal medium and showed that aFGF and KGF were more potent mitogens than EGF, transforming growth factor-alpha, and hepatocyte growth factor. Additionally, basal medium + KGF also allowed progression to a distal alveolar phenotype. We conclude that aFGF and KGF may be important mediators in epithelial-mesenchymal interactions. PMID:8897895

  2. Epithelial expression of keratinocytes growth factor in oral precancer lesions

    PubMed Central

    Jimson, Sudha; Murali, S.; Zunt, Susan L.; Goldblatt, Lawrence I.; Srinivasan, Mythily

    2016-01-01

    Background: Keratinocyte growth factor (KGF) is a potent epithelial mitogen that acts by binding the KGF receptors (KGFRs) expressed on epithelial cells and regulates proliferation and differentiation. The objective of this study was to investigate the expression of KGF in the epithelium in oral precancer. Materials and Methods: Archival tissues of oral submucous fibrosis (SMF) and leukoplakia were assessed for epithelial KGF expression by immunohistochemistry and real-time quantitative polymerase chain reaction. Results: KGF was predominantly expressed in the basal and parabasal cells in the epithelium of SMF tissues. KGF transcript in the epithelial cells increased with increasing severity of epithelial dysplasia in oral leukoplakia. Conclusion: Although widely reported as a product secreted by the mesenchymal cells, our data suggest that the KGF is also expressed in oral epithelial cells much like the expression in ovarian epithelial cells. Based on the localization of KGF in cells at the epithelial mesenchymal junction and that of the reported presence of KGFR in oral keratinocytes, a potential mechanism involving paracrine and autocrine interactions of KGF and KGFR in early stages of oral precancer is postulated. PMID:27274338

  3. Cortactin involvement in the keratinocyte growth factor and fibroblast growth factor 10 promotion of migration and cortical actin assembly in human keratinocytes

    SciTech Connect

    Ceccarelli, Simona; Cardinali, Giorgia; Aspite, Nicaela; Picardo, Mauro; Marchese, Cinzia; Torrisi, Maria Rosaria; Mancini, Patrizia . E-mail: patrizia.mancini@uniroma1.it

    2007-05-15

    Keratinocyte growth factor (KGF/FGF7) and fibroblast growth factor 10 (FGF10/KGF2) regulate keratinocyte proliferation and differentiation by binding to the tyrosine kinase KGF receptor (KGFR). KGF induces keratinocyte motility and cytoskeletal rearrangement, whereas a direct role of FGF10 on keratinocyte migration is not clearly established. Here we analyzed the motogenic activity of FGF10 and KGF on human keratinocytes. Migration assays and immunofluorescence of actin cytoskeleton revealed that FGF10 is less efficient than KGF in promoting migration and exerts a delayed effect in inducing lamellipodia and ruffles formation. Both growth factors promoted phosphorylation and subsequent membrane translocation of cortactin, an F-actin binding protein involved in cell migration; however, FGF10-induced cortactin phosphorylation was reduced, more transient and delayed with respect to that promoted by KGF. Cortactin phosphorylation induced by both growth factors was Src-dependent, while its membrane translocation and cell migration were blocked by either Src and PI3K inhibitors, suggesting that both pathways are involved in KGF- and FGF10-dependent motility. Furthermore, siRNA-mediated downregulation of cortactin inhibited KGF- and FGF10-induced migration. These results indicate that cortactin is involved in keratinocyte migration promoted by both KGF and FGF10.

  4. Expression and modulation of nerve growth factor in murine keratinocytes (PAM 212)

    SciTech Connect

    Tron, V.A.; Coughlin, M.D.; Jang, D.E.; Stanisz, J.; Sauder, D.N. )

    1990-04-01

    Nerve growth factor (NGF) is a polypeptide that is required for normal development and maintenance of the sympathetic and sensory nervous systems. Skin has been shown to contain relatively high amounts of NGF, which is in keeping with the finding that the quantity of NGF in a tissue is proportional to the extent of sympathetic innervation of that organ. Since the keratinocyte, a major cellular constituent of the skin, is known to produce other growth factors and cytokines, our experiments were designed to determine whether keratinocytes are a source of NGF. Keratinocyte-conditioned media from the keratinocyte cell line PAM 212 contained NGF-like activity, approximately 2-3 ng/ml, as detected by the neurite outgrowth assay. Freshly isolated BALB/c keratinocytes contained approximately 0.1 ng/ml. Using a cDNA probe directed against NGF, we demonstrated the presence of a 1.3-kb NGF mRNA in both PAM 212 and BALB/c keratinocytes. Since ultraviolet radiation (UV) is a potentially important modulating factor for cytokines in skin, we examined the effect of UV on NGF mRNA expression. Although UV initially inhibited the expression of keratinocyte NGF mRNA (4 h), by 24 h an induction of NGF mRNA was seen. The NGF signal could also be induced by phorbol esters. Thus, keratinocytes synthesize and express NGF, and its expression is modulated by UVB and phorbol esters.

  5. Modulation of growth and differentiation in normal human keratinocytes by transforming growth factor-beta

    SciTech Connect

    Matsumoto, K.; Hashimoto, K.; Hashiro, M.; Yoshimasa, H.; Yoshikawa, K. )

    1990-10-01

    The effect of transforming growth factor-type beta 1(TGF-beta) on the growth and differentiation of normal human skin keratinocytes cultured in serum-free medium was investigated. TGF-beta markedly inhibited the growth of keratinocytes at the concentrations greater than 2 ng/ml under low Ca2+ conditions (0.1 mM). Growth inhibition was accompanied by changes in cell functions related to proliferation. Remarkable inhibition of DNA synthesis was demonstrated by the decrease of (3H)thymidine incorporation. The decrease of (3H)thymidine incorporation was observed as early as 3 hr after addition of TGF-beta. TGF-beta also decreased c-myc messenger RNA (mRNA) expression 30 min after addition of TGF-beta. This rapid reduction of c-myc mRNA expression by TGF-beta treatment is possibly one of the main factors in the process of TGF-beta-induced growth inhibition of human keratinocytes. Since growth inhibition and induction of differentiation are closely related in human keratinocytes, the growth-inhibitory effect of TGF-beta under high Ca2+ conditions was examined. TGF-beta inhibited the growth of keratinocytes under high Ca2+ conditions in the same manner as under low Ca2+ conditions, suggesting that it is a strong growth inhibitor in both low and high Ca2+ environments. The induction of keratinocyte differentiation was evaluated by measuring involucrin expression and cornified envelope formation: TGF-beta at 20 ng/ml increased involucrin expression from 9.3% to 18.8% under high Ca2+ conditions, while it decreased involucrin expression from 7.0% to 3.3% under low Ca2+ conditions. Cornified envelope formation was modulated in a similar way by addition of TGF-beta: TGF-beta at 20 ng/ml decreased cornified envelope formation by 53% under low Ca2+ conditions, while it enhanced cornified envelope formation by 30.7% under high Ca2+ conditions.

  6. Keratinocyte response to immobilized growth factors for enhanced dermal wound healing

    NASA Astrophysics Data System (ADS)

    Stefonek-Puccinelli, Tracy Jane

    Chronic wounds cost billions of dollars per year to treat and wound care is limited to ineffective and/or expensive options. Chronic wounds are characterized by a failure to reepithelialize, as well as deficiencies in growth factors, such as epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1), normally present during wound healing. Our system described herein begins to tackle the problems associated with designing bioactive materials for chronic wound healing applications. We show that we can induce accelerated keratinocyte migration with photo-immobilized EGF and further control migration speed through the culture of cells on different types of gradient patterns of EGF. We also successfully immobilized IGF-1 while retaining its bioactivity, and further showed it induces directed keratinocyte migration, although not as potently as immobilized EGF. Potential synergy between co-immobilized IGF-1 and EGF was also investigated, although EGF continued to dominate the cellular response, and no significant increase in cell migration was achieved via the addition of IGF-1 to the system. To further understand cellular response to our immobilized growth factors, we investigated keratinocyte signaling and function in response to changes in EGF presentation. It was found that immobilized and soluble EGF can play different, yet complementary, roles in regulating keratinocyte function. Specifically, keratinocytes responded to immobilized EGF with high EGF receptor (EGFR) activation, accompanied by low proliferation and high migratory activity. In contrast, keratinocytes treated with soluble EGF displayed a highly proliferative, rather than migratory, phenotype. We then transitioned our photo-immobilization techniques to materials that may be more suitable as a wound dressing, such as silk fibroin films. Silk fibroin is a natural fiber with many desirable qualities for a biomaterial including high strength and elasticity, biocompatibility, a beta

  7. Endothelin-1 acts as an autocrine growth factor for normal human keratinocytes.

    PubMed

    Tsuboi, R; Sato, C; Shi, C M; Nakamura, T; Sakurai, T; Ogawa, H

    1994-05-01

    Endothelin-1 (ET-1) is an endothelium-derived 21 amino acid vasoconstrictor peptide possessing two intrachain disulfide bridges. Recently it has become evident that isoforms of ET (ET-1, -2, and -3) have a wide range of pharmacological effects in various tissues and act as autocrine/paracrine factors. We demonstrate here that ET-1 is secreted from normal human keratinocytes and may work as an autocrine growth factor through a specific receptor. In this study, human foreskin keratinocytes were cultured in serum-free MCDB 153 medium. Cell growth and [3H] thymidine incorporation in low and high Ca++ concentration media was stimulated by ET-1, -2, and -3 with similar potencies. The strongest response was observed at 10 nM ETs, whereas stimulatory activity was reduced at 100 nM. ETs suppressed keratinocyte differentiation as measured by reactivity with involucrin antibody. Plasminogen activator activity (mainly urokinase) in the medium was also stimulated by the addition of 10 nM ETs. ET-1-like immunoreactivity measured by radioimmunoassay was 1.4 fmol/day/10(6) cells in non-treated condition medium. Among the various cytokines, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 alpha, and transforming growth factor-beta stimulated ET-1 secretion in a dose-dependent manner. The strongest response (ten-fold) was observed upon the addition of 10 ng/ml TNF-alpha. Scatchard plot analysis of [125I] ET-1 binding to keratinocytes revealed the presence of a single class of high affinity receptors (KD 50 pM, 9 x 10(3) sites/cell). Binding was competitively inhibited by the addition of unlabeled ET-1 and -2 with similar affinities and by ET-3 with weaker affinity. ET-1 mRNA expression in keratinocytes was detected by reverse transcription-polymerase chain reaction and was increased by treatment with 10 ng/ml TNF-alpha. These results suggest that ET-1 acts as an autocrine growth factor for keratinocytes through a specific receptor.

  8. Fibroblast Growth Factor-Peptide Improves Barrier Function and Proliferation in Human Keratinocytes After Radiation

    SciTech Connect

    Zhang Kunzhong; Tian Yeping; Yin Liangjie; Zhang Mei; Beck, Lisa A.; Zhang, Bingrong; Okunieff, Paul; Zhang Lurong; Vidyasagar, Sadasivan

    2011-09-01

    Purpose: Epidermal keratinocytes, which can be severely damaged after ionizing radiation (IR), are rapid turnover cells that function as a barrier, protecting the host from pathogenic invasion and fluid loss. We tested fibroblast growth factor-peptide (FGF-P), a small peptide derived from the receptor-binding domain of FGF-2, as a potential mitigator of radiation effects via proliferation and the barrier function of keratinocytes. Methods and Materials: Keratinocytes isolated from neonatal foreskin were grown on transwells. After being exposed to 0, 5, or 10 Gy IR, the cells were treated with a vehicle or FGF-P. The permeability of IR cells was assessed by using transepithelial electrical resistance (TEER) and a paracellular tracer flux of fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) with Ussing chambers. The cell proliferation was measured with yellow tetrazolium salt (MTT) and tritiated thymidine ([{sup 3}H]-TdR) assays. The phosphorylation of extracellular signal-regulated kinases (ERK) was measured in an enzyme-linked immunosorbent (ELISA)-like assay, and the proteins related to tight junctions (TJ) and adherens junctions (AJ) were examined with Western blotting. We used a mouse model to assess the ability of FGF-P to promote the healing of skin {beta} burns created with a strontium applicator. Results: We found (1) FGF-P reduced the permeability of irradiated keratinocytes, as evidenced by increased TEER and decreased diffusion of FITC-BSA, both associated with the regulation of different proteins and levels of TJ and AJ; and (2) FGF-P enhanced the proliferation of irradiated keratinocytes, as evidenced by increased MTT activity and [{sup 3}H]-TdR incorporation, which was associated with activation of the ERK pathway; and (3) FGF-P promoted the healing of skin {beta} burns. Conclusions: FGF-P enhances the barrier function, including up-regulation of TJ proteins, increases proliferation of human keratinocytes, and accelerates the

  9. Arsenite and insulin exhibit opposing effects on epidermal growth factor receptor and keratinocyte proliferative potential

    SciTech Connect

    Patterson, Timothy J.; Rice, Robert H. . E-mail: rhrice@ucdavis.edu

    2007-05-15

    Previous work has suggested that arsenic exposure contributes to skin carcinogenesis by preserving the proliferative potential of human epidermal keratinocytes, thereby slowing the exit of putative target stem cells into the differentiation pathway. To find a molecular basis for this action, present work has explored the influence of arsenite on keratinocyte responses to epidermal growth factor (EGF). The ability of cultured keratinocytes to found colonies upon passaging several days after confluence was preserved by arsenite and EGF in an additive fashion, but neither was effective when the receptor tyrosine kinase activity was inhibited. Arsenite prevented the loss of EGF receptor protein and phosphorylation of tyrosine 1173, preserving its capability to signal. The level of nuclear {beta}-catenin was higher in cells treated with arsenite and EGF in parallel to elevated colony forming ability, and expression of a dominant negative {beta}-catenin suppressed the increase in both colony forming ability and yield of putative stem cells induced by arsenite and EGF. As judged by expression of three genes regulated by {beta}-catenin, this transcription factor had substantially higher activity in the arsenite/EGF-treated cells. Trivalent antimony exhibited the same effects as arsenite. A novel finding is that insulin in the medium induced the loss of EGF receptor protein, which was largely prevented by arsenite exposure.

  10. Chemical allergens stimulate human epidermal keratinocytes to produce lymphangiogenic vascular endothelial growth factor

    SciTech Connect

    Bae, Ok-Nam; Ahn, Seyeon; Jin, Sun Hee; Hong, Soo Hyun; Lee, Jinyoung; Kim, Eun-Sun; Jeong, Tae Cheon; Chun, Young-Jin; Lee, Ai-Young; Noh, Minsoo

    2015-03-01

    Allergic contact dermatitis (ACD) is a cell-mediated immune response that involves skin sensitization in response to contact with various allergens. Angiogenesis and lymphangiogenesis both play roles in the allergic sensitization process. Epidermal keratinocytes can produce vascular endothelial growth factor (VEGF) in response to UV irradiation and during wound healing. However, the effect of haptenic chemical allergens on the VEGF production of human keratinocytes, which is the primary contact site of toxic allergens, has not been thoroughly researched. We systematically investigated whether immune-regulatory cytokines and chemical allergens would lead to the production of VEGF in normal human keratinocytes (NHKs) in culture. VEGF production significantly increased when NHKs were treated with IFNγ, IL-1α, IL-4, IL-6, IL-17A, IL-22 or TNFα. Among the human sensitizers listed in the OECD Test Guideline (TG) 429, we found that CMI/MI, DNCB, 4-phenylenediamine, cobalt chloride, 2-mercaptobenzothiazole, citral, HCA, cinnamic alcohol, imidazolidinyl urea and nickel chloride all significantly upregulated VEGF production in NHKs. In addition, common human haptenic allergens such as avobenzone, formaldehyde and urushiol, also induced the keratinocyte-derived VEGF production. VEGF upregulation by pro-inflammatory stimuli, IFNγ, DNCB or formaldehyde is preceded by the production of IL-8, an acute inflammatory phase cytokine. Lymphangiogenic VEGF-C gene transcription was significantly increased when NHKs were treated with formaldehyde, DNCB or urushiol, while transcription of VEGF-A and VEGF-B did not change. Therefore, the chemical allergen-induced VEGF upregulation is mainly due to the increase in lymphangiogenic VEGF-C transcription in NHKs. These results suggest that keratinocyte-derived VEGF may regulate the lymphangiogenic process during the skin sensitization process of ACD. - Highlights: • Pro-inflammatory cytokines induced VEGF production in normal human

  11. Immobilized epidermal growth factor stimulates persistent, directed keratinocyte migration via activation of PLCγ1.

    PubMed

    Kim, Chloe S; Mitchell, Isaiah P; Desotell, Anthony W; Kreeger, Pamela K; Masters, Kristyn S

    2016-07-01

    Epidermal growth factor (EGF) is a critical element in dermal repair, but EGF-containing wound dressings have not been successful clinically. However, these dressings have delivered only soluble EGF, and the native environment provides both soluble and matrix-bound EGF. To address our hypothesis that tethered EGF can stimulate cell behaviors not achievable with soluble EGF, we examined single-cell movement and signaling in human immortalized HaCaT keratinocytes treated with soluble or immobilized EGF. Although both EGF treatments increased collective sheet displacement and individual cell speed, only cells treated with immobilized EGF exhibited directed migration, as well as 2-fold greater persistence compared with soluble EGF. Immunofluorescence showed altered EGF receptor (EGFR) trafficking, where EGFR remained membrane-localized in the immobilized EGF condition. Cells treated with soluble EGF demonstrated higher phosphorylated ERK1/2, and cells on immobilized EGF exhibited higher pPLCγ1, which was localized at the leading edge. Treatment with U0126 inhibited migration in both conditions, demonstrating that ERK1/2 activity was necessary but not responsible for the observed differences. In contrast, PLCγ1 inhibition with U73122 significantly decreased persistence on immobilized EGF. Combined, these results suggest that immobilized EGF increases collective keratinocyte displacement via an increase in single-cell migration persistence resulting from altered EGFR trafficking and PLCγ1 activation.-Kim, C. S., Mitchell, I. P., Desotell, A. W., Kreeger, P. K., Masters, K. S. Immobilized epidermal growth factor stimulates persistent, directed keratinocyte migration via activation of PLCγ1.

  12. Immobilized epidermal growth factor stimulates persistent, directed keratinocyte migration via activation of PLCγ1.

    PubMed

    Kim, Chloe S; Mitchell, Isaiah P; Desotell, Anthony W; Kreeger, Pamela K; Masters, Kristyn S

    2016-07-01

    Epidermal growth factor (EGF) is a critical element in dermal repair, but EGF-containing wound dressings have not been successful clinically. However, these dressings have delivered only soluble EGF, and the native environment provides both soluble and matrix-bound EGF. To address our hypothesis that tethered EGF can stimulate cell behaviors not achievable with soluble EGF, we examined single-cell movement and signaling in human immortalized HaCaT keratinocytes treated with soluble or immobilized EGF. Although both EGF treatments increased collective sheet displacement and individual cell speed, only cells treated with immobilized EGF exhibited directed migration, as well as 2-fold greater persistence compared with soluble EGF. Immunofluorescence showed altered EGF receptor (EGFR) trafficking, where EGFR remained membrane-localized in the immobilized EGF condition. Cells treated with soluble EGF demonstrated higher phosphorylated ERK1/2, and cells on immobilized EGF exhibited higher pPLCγ1, which was localized at the leading edge. Treatment with U0126 inhibited migration in both conditions, demonstrating that ERK1/2 activity was necessary but not responsible for the observed differences. In contrast, PLCγ1 inhibition with U73122 significantly decreased persistence on immobilized EGF. Combined, these results suggest that immobilized EGF increases collective keratinocyte displacement via an increase in single-cell migration persistence resulting from altered EGFR trafficking and PLCγ1 activation.-Kim, C. S., Mitchell, I. P., Desotell, A. W., Kreeger, P. K., Masters, K. S. Immobilized epidermal growth factor stimulates persistent, directed keratinocyte migration via activation of PLCγ1. PMID:27025961

  13. Keratinocyte growth factor functions in epithelial induction during seminal vesicle development.

    PubMed Central

    Alarid, E T; Rubin, J S; Young, P; Chedid, M; Ron, D; Aaronson, S A; Cunha, G R

    1994-01-01

    Development of the seminal vesicle (SV) is elicited by androgens and is dependent on epithelial-mesenchymal interactions. Androgenic signal transmission from the androgen-receptor-positive mesenchyme to the epithelium has been postulated to involve paracrine factors. Keratinocyte growth factor (KGF), a member of the fibroblast growth factor family, is produced by stromal/mesenchymal cells and acts specifically on epithelial cells. The KGF transcript was detected by reverse transcription-polymerase chain reaction in newborn mouse SVs and by Northern blot analysis of RNA from cultured neonatal SV mesenchymal cells. Newborn SVs placed in organ culture undergo androgen-dependent growth and differentiation. Addition of a KGF-neutralizing monoclonal antibody to this system caused striking inhibition of both SV growth and branching morphogenesis. This inhibition was due to a decline in epithelial proliferation and differentiation, as the mesenchymal layer was not affected by anti-KGF treatment. When KGF (100 ng/ml) was substituted for testosterone in the culture medium, SV growth was approximately 50% that observed with an optimal dose of testosterone (10(-7) M). All of these findings suggest that KGF is present during a time of active SV morphogenesis and functions as an important mediator of androgen-dependent development. Images PMID:8302834

  14. Keratinocyte growth factor and hepatocyte growth factor/scatter factor are heparin-binding growth factors for alveolar type II cells in fibroblast-conditioned medium.

    PubMed Central

    Panos, R J; Rubin, J S; Csaky, K G; Aaronson, S A; Mason, R J

    1993-01-01

    Epithelial-mesenchymal interactions mediate aspects of normal lung growth and development and are important in the restoration of normal alveolar architecture after lung injury. To determine if fibroblasts are a source of soluble growth factors for alveolar type II cells, we investigated the effect of fibroblast-conditioned medium (CM) on alveolar type II cell DNA synthesis. Serum-free CM from confluent adult human lung fibroblasts was concentrated fivefold by lyophilization. Type II cells were isolated from adult rats by elastase dissociation and incubated with [3H]thymidine and varying dilutions of concentrated CM and serum from day 1 to 3 of culture. Stimulation of type II cell DNA synthesis by fibroblast-CM was maximal after 48 h of conditioning and required the presence of serum. The activity of the CM was eliminated by boiling and by treatment with trypsin, pepsin, or dithiothreitol and was additive with saturating concentrations of acidic fibroblast growth factor, epidermal growth factor, and insulin. The growth factor activity bound to heparin-Sepharose and was eluted with 0.6 and 1.0 M NaCl. Neutralizing antibody studies demonstrated that the primary mitogens isolated in the 0.6 and 1.0 M NaCl fractions were keratinocyte growth factor (KGF, fibroblast growth factor 7) and hepatocyte growth factor/scatter factor (HGF/SF), respectively. HGF/SF was demonstrated in the crude CM and KGF was detected in the 0.6 M NaCl eluent by immunoblotting. Northern blot analysis confirmed that the lung fibroblasts expressed both KGF and HGF/SF transcripts. Human recombinant KGF and HGF/SF induced a concentration- and serum-dependent increase in rat alveolar type II cell DNA synthesis. We conclude that adult human lung fibroblasts produce at least two soluble heparin-binding growth factors, KGF and HGF/SF, which promote DNA synthesis and proliferation of rat alveolar type II cells in primary culture. KGF and HGF/SF may be important stimuli for alveolar type II cell

  15. A role for the perlecan protein core in the activation of the keratinocyte growth factor receptor.

    PubMed Central

    Ghiselli, G; Eichstetter, I; Iozzo, R V

    2001-01-01

    Perlecan, a widespread heparan sulphate (HS) proteoglycan, is directly involved in the storing of angiogenic growth factors, mostly members of the fibroblast growth factor (FGF) gene family. We have previously shown that antisense targeting of the perlecan gene causes a reduced growth and responsiveness to FGF7 [also known as keratinocyte growth factor (KGF)] in human cancer cells, and that the perlecan protein core interacts specifically with FGF7. In the present paper, we have investigated human colon carcinoma cells in which the perlecan gene was disrupted by targeted homologous recombination. After screening over 1000 clones, we obtained two clones heterozygous for the null mutation with no detectable perlecan, indicating that the other allele was non-functioning. The perlecan-deficient cells grew more slowly, did not respond to FGF7 with or without the addition of heparin, and were less tumorigenic than control cells. Paradoxically, the perlecan-deficient cells displayed increased FGF7 surface binding. However, the perlecan protein core was required for functional activation of the KGF receptor and downstream signalling. Because heparin could not substitute for perlecan, the HS chains are not critical for FGF7-mediated signalling in this cell system. These results provide the first genetic evidence that the perlecan protein core is a molecular entity implicated in FGF7 binding and activation of its receptor. PMID:11563979

  16. Noncovalent PEGylation by polyanion complexation as a means to stabilize keratinocyte growth factor-2 (KGF-2).

    PubMed

    Khondee, Supang; Olsen, Christopher M; Zeng, Yuhong; Middaugh, C Russell; Berkland, Cory

    2011-11-14

    Repifermin, a truncated form of fibroblast growth factor-10 (FGF-10) also known as keratinocyte growth factor-2 (KGF-2), is a heparin-binding protein with potent regenerative properties. The protein unfolds and aggregates at relatively low temperature (~37 °C). Electrostatic interactions between polyanions and several FGFs have been reported to enhance the thermal stability of these proteins. Polyethylene glycol (PEG) was grafted to the polyanions pentosan polysulfate (PPS) and dextran sulfate (DS) as an alternative means to stabilize and noncovalently PEGylate KGF-2. Physical characteristics of KGF-2:polyanion-PEG complexes were examined using a variety of methods including circular dichroism (CD), intrinsic tryptophan fluorescence, differential scanning calorimetry, and dynamic light scattering. When compared to KGF-2 alone, subtle changes in CD spectra and fluorescence emission maxima were found when KGF-2 was formulated with the synthetic PEG-polyanions. Highly PEGylated polyanions (DS-PEG5) did not bind KGF-2 as well as conjugates with fewer PEG chains. The molecular weight of PEG did not have a noticeable effect on KGF-2 binding to the various PEG-polyanion conjugates. At optimal molar ratios, PPS-PEG and DS-PEG conjugates were able to stabilize KGF-2 by increasing the melting temperature by approximately 9-17 °C. Thus, polyanion-PEG conjugates improved the stability of KGF-2 and also offered a new electrostatic PEGylation scheme that may be extrapolated to other heparin-binding proteins.

  17. Regulation of vascular endothelial growth factor expression in human keratinocytes by retinoids.

    PubMed

    Diaz, B V; Lenoir, M C; Ladoux, A; Frelin, C; Démarchez, M; Michel, S

    2000-01-01

    Vascular endothelial growth factor (VEGF) is overexpressed in hyperproliferative diseases, such as psoriasis and cancers, which are characterized by increased angiogenesis. Experimentally, VEGF overexpression can be induced by the treatment of cell cultures and biological tissues with phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA). Using normal human keratinocytes in conventional cultures and skin grafted onto nude mice in vivo, we show that retinoids can inhibit TPA-mediated VEGF gene induction at the transcriptional level. Because retinoids are biologically active either by interacting with the nuclear retinoic acid receptors or by interfering with the activator protein 1 (AP1) transcription factor, we studied the effect of the retinoic acid derivative CD 2409, which exhibits strong anti-AP1 activity but does not bind to the known retinoic acid receptors in vitro. The results demonstrate that the inhibition of VEGF expression by retinoids only depends on their anti-AP1 activity and does not require gene transactivation via retinoic acid response elements. Because the VEGF promoter contains four potential AP1 binding sites, we used different promoter constructs to identify the functional site responsible for TPA induction and retinoid inhibition. This site turned out to be localized at position -621 of the 5' flanking region of the VEGF gene.

  18. Keratinocyte growth factor-2 is protective in lipopolysaccharide-induced acute lung injury in rats.

    PubMed

    Tong, Lin; Bi, Jing; Zhu, Xiaodan; Wang, Guifang; Liu, Jie; Rong, Linyi; Wang, Qin; Xu, Nuo; Zhong, Ming; Zhu, Duming; Song, Yuanlin; Bai, Chunxue

    2014-09-15

    Keratinocyte growth factor-2 (KGF-2) plays a key role in lung development, but its role in acute lung injury has not been well characterized. Lipopolysaccharide instillation caused acute lung injury, which significantly elevated lung wet-to-dry weight ratio, protein and neutrophils in bronchoalveolar lavage fluid (BALF), inhibited surfactant protein A and C expression in lung tissue, and increased pathological injury. Pretreatment with KGF-2 improved the above lung injury parameters, partially restored surfactant protein A and C expression, and KGF-2 given 2-3 days before LPS challenge showed maximum lung injury improvement. Pretreatment with KGF-2 also markedly reduced the levels of TNF-α, MIP-2, IL-1β and IL-6 in BALF and the levels of IL-1β and IL-6 in lung tissue. Histological analysis showed there was increased proliferation of alveolar type II epithelial cells in lung parenchyma, which reached maximal 2 days after KGF-2 instillation. Intratracheal administration of KGF-2 attenuates lung injury induced by LPS, suggesting KGF-2 may be potent in the intervention of acute lung injury.

  19. Galectin-3 regulates intracellular trafficking of epidermal growth factor receptor through Alix and promotes keratinocyte migration

    PubMed Central

    Liu, Wei; Hsu, Daniel K.; Chen, Huan-Yuan; Yang, Ri-Yao; Carraway, Kermit L.; Isseroff, Roslyn R.; Liu, Fu-Tong

    2012-01-01

    The epidermal growth factor receptor (EGFR)-mediated signaling pathways are important in a variety of cellular processes, including cell migration and wound re-epithelialization. Intracellular trafficking of EGFR is critical for maintaining EGFR surface expression. Galectin-3, a member of an animal lectin family, has been implicated in a number of physiological and pathological processes. Through studies of galectin-3-deficient mice and cells isolated from these mice, we demonstrated that absence of galectin-3 impairs keratinocyte migration and skin wound re-epithelialization. We have linked this pro-migratory function to a crucial role of cytosolic galectin-3 in controlling intracellular trafficking and cell surface expression of EGFR after EGF stimulation. Without galectin-3, the surface levels of EGFR are dramatically reduced and the receptor accumulates diffusely in the cytoplasm. This is associated with reduced rates of both endocytosis and recycling of the receptor. We have provided evidence that this novel function of galectin-3 may be mediated through interaction with its binding partner Alix, which is a protein component of the endosomal sorting complex required for transport (ESCRT) machinery. Our results suggest that galectin-3 is potentially a critical regulator of a number of important cellular responses through its intracellular control of trafficking of cell surface receptors. PMID:22785133

  20. Strengthening the Skin with Topical Delivery of Keratinocyte Growth Factor-1 Using a Novel DNA Plasmid

    PubMed Central

    Dou, Chunqing; Lay, Frank; Ansari, Amir Mehdi; Rees, Donald J; Ahmed, Ali Karim; Kovbasnjuk, Olga; Matsangos, Aerielle E.; Du, Junkai; Hosseini, Sayed Mohammad; Steenbergen, Charles; Fox-Talbot, Karen; Tabor, Aaron T.; Williams, James A; Liu, Lixin; Marti, Guy P; Harmon, John W

    2014-01-01

    Fragile skin, susceptible to decubitus ulcers and incidental trauma, is a problem particularly for the elderly and for those with spinal cord injury. Here, we present a simple approach to strengthen the skin by the topical delivery of keratinocyte growth factor-1 (KGF-1) DNA. In initial feasibility studies with the novel minimalized, antibiotic-free DNA expression vector, NTC8385-VA1, the reporter genes luciferase and enhanced green fluorescent protein were delivered. Transfection was documented when luciferase expression significantly increased after transfection. Microscopic imaging of enhanced green fluorescent protein–transfected skin showed green fluorescence in hair follicles, hair shafts, and dermal and superficial epithelial cells. With KGF-1 transfection, KGF-1 mRNA level and protein production were documented with quantitative reverse transcriptase–polymerase chain reaction and immunohistochemistry, respectively. Epithelial thickness of the transfected skin in the KGF group was significantly increased compared with the control vector group (26 ± 2 versus 16 ± 4 µm) at 48 hours (P = 0.045). Dermal thickness tended to be increased in the KGF group (255 ± 36 versus 162 ± 16 µm) at 120 hours (P = 0.057). Biomechanical assessment showed that the KGF-1–treated skin was significantly stronger than control vector–transfected skin. These findings indicate that topically delivered KGF-1 DNA plasmid can increase epithelial thickness and strength, demonstrating the potential of this approach to restore compromised skin. PMID:24434934

  1. Loss of p53 induces epidermal growth factor receptor promoter activity in normal human keratinocytes

    PubMed Central

    Bheda, A; Creek, KE; Pirisi, L

    2008-01-01

    Overexpression of the epidermal growth factor receptor (EGFR) in human papillomavirus type 16-immortalized human keratinocytes (HKc) is caused by the viral oncoprotein E6, which targets p53 for degradation. We have previously observed that expression of p53 RNAi in normal HKc is associated with an increase in EGFR mRNA and protein. We now report that p53 RNAi induces EGFR promoter activity up to approximately 10-fold in normal HKc, and this effect does not require intact p53 binding sites on the EGFR promoter. Exogenous wild-type p53 inhibits the EGFR promoter at low levels, and activates it at higher concentrations. Yin Yang 1 (YY1), which negatively regulates p53, induces EGFR promoter activity, and this effect is augmented by p53 RNAi. Intact p53 binding sites on the EGFR promoter are not required for activation by YY1. In addition, Sp1 and YY1 synergistically induce the EGFR promoter in normal HKc, indicating that Sp1 may recruit YY1 as a co-activator. Wild-type p53 suppressed Sp1- and YY1-mediated induction of the EGFR promoter. We conclude that acute loss of p53 in normal HKc induces EGFR expression bya mechanism that involves YY1 and Sp1 and does not require p53 binding to the EGFR promoter. PMID:18391986

  2. Kinetics and regulation of human keratinocyte stem cell growth in short-term primary ex vivo culture. Cooperative growth factors from psoriatic lesional T lymphocytes stimulate proliferation among psoriatic uninvolved, but not normal, stem keratinocytes.

    PubMed Central

    Bata-Csorgo, Z; Hammerberg, C; Voorhees, J J; Cooper, K D

    1995-01-01

    Flow cytometric analysis of primary ex vivo keratinocyte cultures demonstrated that stem cells, (beta 1 integrin+, keratin 1/keratin 10 [K1/K10-], proliferating cell nuclear antigen [PCNA-] [Bata-Csorgo, Zs., C. Hammerberg, J. J. Voorhees, and K. D. Cooper. 1993. J. Exp. Med. 178:1271-1281]) establish such cultures. This methodology also enabled the quantitation of synchronized recruitment of these cells from G0 into G1 of the cell cycle (PCNA expression), which preceded bright beta 1 integrin expression. (beta 1 integrinbright expression has been shown to be a characteristic feature of keratinocyte stem cells in culture (Jones, P. H., and F. M. Watt. 1993. Cell. 73:713-724). Using the above assay, we determined whether lesional T lymphocytes in psoriasis could be directly responsible for the induction of the stem cell hyperproliferation that is characteristic of this disease. Indeed, CD4+ T lymphocytes, cloned from lesional psoriatic skin and stimulated by immobilized anti-CD3 plus fibronectin, promoted psoriatic uninvolved keratinocyte stem cell proliferation via soluble factors. This induction appeared to be through accelerated recruitment of stem cells from their quiescent state (G0) into cell cycle. By contrast, normal keratinocyte stem cells exhibited no such growth stimulation. Supernatants exhibiting growth induction all contained high levels of GM-CSF and gamma-IFN, low IL-3 and TNF-alpha, and variable IL-4. Only anti-gamma-IFN antibody was able to neutralize growth stimulatory activity of the supernatants on psoriatic uninvolved keratinocyte stem cells. However, because recombinant gamma-IFN alone inhibited growth in this assay, these data suggest that, in psoriasis, gamma-IFN acts cooperatively with other growth factors in the immune induction of cell cycle progression by the normally quiescent stem cell keratinocytes. Images PMID:7529261

  3. Correlation between the 1.6 A crystal structure and mutational analysis of keratinocyte growth factor.

    PubMed Central

    Osslund, T. D.; Syed, R.; Singer, E.; Hsu, E. W.; Nybo, R.; Chen, B. L.; Harvey, T.; Arakawa, T.; Narhi, L. O.; Chirino, A.; Morris, C. F.

    1998-01-01

    A comprehensive deletion, mutational, and structural analysis of the native recombinant keratinocyte growth factor (KGF) polypeptide has resulted in the identification of the amino acids responsible for its biological activity. One of these KGF mutants (delta23KGF-R144Q) has biological activity comparable to the native protein, and its crystal structure was determined by the multiple isomorphous replacement plus anomalous scattering method (MIRAS). The structure of KGF reveals that it folds into a beta-trefoil motif similar to other members of fibroblast growth factor (FGF) family whose structures have been resolved. This fold consists of 12 anti-parallel beta-strands in which three pairs of the strands form a six-stranded beta-barrel structure and the other three pairs of beta-strands cap the barrel with hairpin triplets forming a triangular array. KGF has 10 well-defined beta strands, which form five double-stranded anti-parallel beta-sheets. A sixth poorly defined beta-strand pair is in the loop between residues 133 and 144, and is defined by only a single hydrogen bond between the two strands. The KGF mutant has 10 additional ordered amino terminus residues (24-33) compared to the other FGF structures, which are important for biological activity. Based on mutagenesis, thermal stability, and structural data we postulate that residues TRP125, THR126, and His127 predominantly confer receptor binding specificity to KGF. Additionally, residues GLN152, GLN138, and THR42 are implicated in heparin binding. The increased thermal stability of delta23KGF-R144Q can structurally be explained by the additional formation of hydrogen bonds between the GLN side chain and a main-chain carbonyl on an adjoining loop. The correlation of the structure and biochemistry of KGF provides a framework for a rational design of this potentially important human therapeutic. PMID:10082365

  4. Platelet-released growth factors induce the antimicrobial peptide human beta-defensin-2 in primary keratinocytes.

    PubMed

    Bayer, Andreas; Lammel, Justus; Rademacher, Franziska; Groß, Justus; Siggelkow, Markus; Lippross, Sebastian; Klüter, Tim; Varoga, Deike; Tohidnezhad, Mersedeh; Pufe, Thomas; Cremer, Jochen; Gläser, Regine; Harder, Jürgen

    2016-06-01

    Platelet-released growth factors (PRGF) and its related clinically used formulations [e.g. Vivostat platelet-rich fibrin (PRF(®) )] are thrombocyte concentrate lysates that support healing of chronic, hard-to-heal and infected wounds. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide expressed in human keratinocytes exhibiting potent antimicrobial activity against wound-related bacteria. In this study, we analysed the influence of PRGF on hBD-2 expression in human primary keratinocytes and the influence of Vivostat PRF(®) on hBD-2 expression in experimentally generated skin wounds in vivo. Treatment of primary keratinocytes with PRGF caused a significant increase in hBD-2 gene and protein expressions in a concentration- and time-dependent manner. The use of blocking antibodies revealed that the PRGF-mediated hBD-2 induction was partially mediated by the epidermal growth factor receptor and the interleukin-6 receptor (IL-6R). Luciferase gene reporter assays indicated that the hBD-2 induction through PRGF required activation of the transcription factor activator protein 1 (AP-1), but not of NF-kappaB. In concordance with these cell culture data, Vivostat PRF(®) induced hBD-2 expression when applied to experimentally generated skin wounds. Together, our results indicate that the induction of hBD-2 by thrombocyte concentrate lysates can contribute to the observed beneficial effects in the treatment of chronic and infected wounds. PMID:26843467

  5. Selective inhibition of growth-related gene expression in murine keratinocytes by transforming growth factor beta.

    PubMed Central

    Coffey, R J; Bascom, C C; Sipes, N J; Graves-Deal, R; Weissman, B E; Moses, H L

    1988-01-01

    Transforming growth factor beta (TGF beta) is a potent inhibitor of epithelial cell proliferation. A nontumorigenic epidermal growth factor (EGF)-dependent epithelial cell line, BALB/MK, is reversibly growth arrested by TGF beta. TGF beta will also abrogate EGF-stimulated mitogenesis of quiescent BALB/MK cells. Increased levels of calcium (greater than 1.0 mM) will induce differentiation in BALB/MK cells; in contrast, TGF beta-mediated growth inhibition does not result in induction of terminal differentiation. In the present study, the effects of TGF beta and calcium on growth factor-inducible gene expression were examined. TGF beta markedly decreased c-myc and KC gene expression in rapidly growing BALB/MK cells and reduced the EGF induction of c-myc and KC in a quiescent population of cells. TGF beta exerted its control over c-myc expression at a posttranscriptional level, and this inhibitory effect was dependent on protein synthesis. TGF beta had no effect on c-fos gene expression, whereas 1.5 mM calcium attenuated EGF-induced c-fos expression in quiescent cells. Expression of beta-actin, however, was slightly increased in both rapidly growing and EGF-restimulated quiescent BALB/MK cells treated with TGF beta. Thus, in this system, TGF beta selectively reduced expression of certain genes associated with cell proliferation (c-myc and KC), and at least part of the TGF beta effect was at a posttranscriptional level. Images PMID:2463471

  6. Efficacy of keratinocyte growth factor (palifermin) for the treatment of caustic esophageal burns.

    PubMed

    Numanoğlu, Kemal Varim; Tatli, Duygu; Bektaş, Sibel; Er, Ebubekir

    2014-10-01

    Current treatment strategies against the development of corrosive esophageal strictures remain unsatisfactory. Thus, the aim of the present study was to investigate the efficacy of keratinocyte growth factor, in the form of palifermin, for the prevention of stricture development following esophageal caustic injuries in a rat model. A total of 32 female Wistar albino rats were divided into four groups, which included the control (C), burn (B), steroid (S) and steroid plus palifermin (S/P) groups. An experimental corrosive esophageal burn model was established in the B, S and S/P groups. Weight gain was recorded and histopathological evaluation was performed for each group. Weight gain in the S and B groups was compared with the control group and statistically significant differences were observed. In addition, statistically significant differences in weight gain were observed between the S/P group and the B group. Histopathologically, statistically significant differences were identified with regard to submucosal collagen deposition, muscularis mucosa and tunica muscularis damage when comparing the B group with the C group. In addition, statistically significant differences were observed when comparing the S and S/P groups with the B group. Furthermore, significant submucosal collagen deposition and tunica muscularis damage were observed in the S group when compared with the S/P group. The stenosis indexes in the C and S groups were significantly lower compared with the B group. In addition, the stenosis index in the S/P group was significantly lower compared with the S group. To the best of our knowledge, the present study is the first to investigate the effect of palifermin on corrosive esophageal burns. The addition of palifermin to the corrosive esophageal burn standard treatment regimen was found to reduce the degree of fibrosis and ameliorate histopathological damage in an experimental model of corrosive esophagitis in rats. PMID:25187801

  7. Tailored delivery of active keratinocyte growth factor from biodegradable polymer formulations.

    PubMed

    Cho, Eun Jeong; Tao, Zunyu; Tang, Ying; Tehan, Elizabeth C; Bright, Frank V; Hicks, Wesley L; Gardella, Joseph A; Hard, Robert

    2003-08-01

    We report the results of a high throughput screening campaign that is aimed to develop a biodegradable polymer-based formulation to deliver active keratinocyte growth factor (KGF) and provide a means to tune the KGF delivery rate. A statistical design strategy was used to prepare and screen a series of polymer blends that were composed of poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and the surfactant sodium bis(ethylhexyl)sulfosuccinate (Aerosol-OT, AOT). Chloroform was the solvent. Our high throughput screening method used a two-tiered assessment strategy. At Level 1, we identified "lead" KFG-loaded formulations that exhibited KGF emission spectra that were the most similar to the native KGF spectrum recorded in buffer. At Level 2, we used steady-state emission and a homogeneous polarization immunoassay strategy to determine the concentration of total and active KGF, respectively, liberated from the lead formulations during biodegradation. After preparing and screening 2500 formulations, we identified several viable, lead formulations. An analysis of the data showed that the combination of PLA, PGA, and AOT were important to yield a high fraction of active KGF upon release from the formulation; no combination of any two together produced an effect as good as the ternary formulation. The optimum formulations that yielded the highest fraction of active KGF upon release had the following general features: PLA/PGA (w/w) near unity, AOT loading of 100-200 mM, water/AOT mole ratio of 10-20, and a pH between 6 and 8. PLA alone cast from chloroform delivered KGF, but that KGF did not bind to anti-KGF antibodies (i.e., it was inactive). We can tune the KGF release kinetics by more than two orders of magnitude while maintaining the KGF activity upon liberation from the formulation by adjusting the PLA molecular weight.

  8. Keratinocyte Growth Factor Combined with a Sodium Hyaluronate Gel Inhibits Postoperative Intra-Abdominal Adhesions

    PubMed Central

    Wei, Guangbing; Zhou, Cancan; Wang, Guanghui; Fan, Lin; Wang, Kang; Li, Xuqi

    2016-01-01

    Postoperative intra-abdominal adhesion is a very common complication after abdominal surgery. One clinical problem that remains to be solved is to identify an ideal strategy to prevent abdominal adhesions. Keratinocyte growth factor (KGF) has been proven to improve the proliferation of mesothelial cells, which may enhance fibrinolytic activity to suppress postoperative adhesions. This study investigated whether the combined administration of KGF and a sodium hyaluronate (HA) gel can prevent intra-abdominal adhesions by improving the orderly repair of the peritoneal mesothelial cells. The possible prevention mechanism was also explored. The cecum wall and its opposite parietal peritoneum were abraded after laparotomy to induce intra-abdominal adhesion formation. Animals were randomly allocated to receive topical application of HA, KGF, KGF + HA, or normal saline (Control). On postoperative day 7, the adhesion score was assessed with a visual scoring system. Masson’s trichrome staining, picrosirius red staining and hydroxyproline assays were used to assess the magnitude of adhesion and tissue fibrosis. Cytokeratin, a marker of the mesothelial cells, was detected by immunohistochemistry. The levels of tissue plasminogen activator (tPA), interleukin-6 (IL-6), and transforming growth factor β1 (TGF-β1) in the abdominal fluid were determined using enzyme-linked immunosorbent assays (ELISAs). Western blotting was performed to examine the expression of the TGF-β1, fibrinogen and α-smooth muscle actin (α-SMA) proteins in the rat peritoneal adhesion tissue. The combined administration of KGF and HA significantly reduced intra-abdominal adhesion formation and fibrin deposition and improved the orderly repair of the peritoneal mesothelial cells in the rat model. Furthermore, the combined administration of KGF and HA significantly increased the tPA levels but reduced the levels of IL-6, tumor necrosis factor α (TNF-α) and TGF-β1 in the abdominal fluid. The expression

  9. Keratinocyte Growth Factor Combined with a Sodium Hyaluronate Gel Inhibits Postoperative Intra-Abdominal Adhesions.

    PubMed

    Wei, Guangbing; Zhou, Cancan; Wang, Guanghui; Fan, Lin; Wang, Kang; Li, Xuqi

    2016-01-01

    Postoperative intra-abdominal adhesion is a very common complication after abdominal surgery. One clinical problem that remains to be solved is to identify an ideal strategy to prevent abdominal adhesions. Keratinocyte growth factor (KGF) has been proven to improve the proliferation of mesothelial cells, which may enhance fibrinolytic activity to suppress postoperative adhesions. This study investigated whether the combined administration of KGF and a sodium hyaluronate (HA) gel can prevent intra-abdominal adhesions by improving the orderly repair of the peritoneal mesothelial cells. The possible prevention mechanism was also explored. The cecum wall and its opposite parietal peritoneum were abraded after laparotomy to induce intra-abdominal adhesion formation. Animals were randomly allocated to receive topical application of HA, KGF, KGF + HA, or normal saline (Control). On postoperative day 7, the adhesion score was assessed with a visual scoring system. Masson's trichrome staining, picrosirius red staining and hydroxyproline assays were used to assess the magnitude of adhesion and tissue fibrosis. Cytokeratin, a marker of the mesothelial cells, was detected by immunohistochemistry. The levels of tissue plasminogen activator (tPA), interleukin-6 (IL-6), and transforming growth factor β1 (TGF-β1) in the abdominal fluid were determined using enzyme-linked immunosorbent assays (ELISAs). Western blotting was performed to examine the expression of the TGF-β1, fibrinogen and α-smooth muscle actin (α-SMA) proteins in the rat peritoneal adhesion tissue. The combined administration of KGF and HA significantly reduced intra-abdominal adhesion formation and fibrin deposition and improved the orderly repair of the peritoneal mesothelial cells in the rat model. Furthermore, the combined administration of KGF and HA significantly increased the tPA levels but reduced the levels of IL-6, tumor necrosis factor α (TNF-α) and TGF-β1 in the abdominal fluid. The expression

  10. Granulocyte/macrophage colony-stimulating factor is an intrinsic keratinocyte-derived growth factor for human melanocytes in UVA-induced melanosis.

    PubMed

    Imokawa, G; Yada, Y; Kimura, M; Morisaki, N

    1996-01-15

    Recently we demonstrated that endothelins secreted from human keratinocytes act as intrinsic mitogens and melanogens for human melanocytes in UVB-induced melanosis. We show here that UVA-induced melanosis is associated with other keratinocyte-derived growth factors, secretion of which is specifically stimulated after exposure of human keratinocytes to UVA. Medium conditioned by UVA-exposed human keratinocytes elicited a significant increase in DNA synthesis by cultured human melanocytes in a UVA dose-dependent manner. Analysis of endothelin-1 and interleukin (IL)-1 alpha in the conditioned medium by ELISA, both of which are major keratinocyte-derived cytokines involved in UVB-associated melanocyte activation, revealed that UVA exposure did not cause human keratinocytes to stimulate the secretion of the two cytokines. In contrast, the levels of several other cytokines such as IL-6, IL-8 and granulocyte/macrophage colony-stimulating factor (GM-CSF) were significantly increased in the conditioned medium of human keratinocytes after exposure to UVA at a dose of 1.0 J/cm2. The gel chromatographic profile of UVA-exposed keratinocyte-conditioned medium demonstrated that there were two factors (P-1 and P-2) with molecular masses of approx. 20 and 1 kDa respectively that stimulate DNA synthesis in human melanocytes, and the larger species (P-1) also increased melanization as assessed by [14C]thiouracil incorporation. Quantitative analysis of cytokines in chromatographic fractions by ELISA revealed the P-1 fraction to be consistent with the molecular mass profile of GM-CSF. Furthermore the stimulatory effect of the P-1 fraction on DNA synthesis in human melanocytes was neutralized by antibodies to GM-CSF, but not to basic fibroblast growth factor or stem cell factor. Binding and proliferation assays with recombinant GM-CSF demonstrated that human melanocytes possess specific binding sites for GM-CSF(Kd 2.11 nM; binding sites, 2.5-3.5 x 10(4) per cell), and recombinant GM

  11. Growth factors in porcine full and partial thickness burn repair. Differing targets and effects of keratinocyte growth factor, platelet-derived growth factor-BB, epidermal growth factor, and neu differentiation factor.

    PubMed Central

    Danilenko, D. M.; Ring, B. D.; Tarpley, J. E.; Morris, B.; Van, G. Y.; Morawiecki, A.; Callahan, W.; Goldenberg, M.; Hershenson, S.; Pierce, G. F.

    1995-01-01

    The topical application of recombinant growth factors such as epidermal growth factor, platelet-derived growth factor-BB homodimer (rPDGF-BB), keratinocyte growth factor (rKGF), and neu differentiation factor has resulted in significant acceleration of healing in several animal models of wound repair. In this study, we established highly reproducible and quantifiable full and deep partial thickness porcine burn models in which burns were escharectomized 4 or 5 days postburn and covered with an occlusive dressing to replicate the standard treatment in human burn patients. We then applied these growth factors to assess their efficacy on several parameters of wound repair: extracellular matrix and granulation tissue production, percent reepithelialization, and new epithelial area. In full thickness burns, only rPDGF-BB and the combination of rPDGF-BB and rKGF induced significant changes in burn repair. rPDGF-BB induced marked extracellular matrix and granulation tissue production (P = 0.013) such that the burn defect was filled within several days of escharectomy, but had no effect on new epithelial area or reepithelialization. The combination of rPDGF-BB and rKGF in full thickness burns resulted in a highly significant increase in extracellular matrix and granulation tissue area (P = 0.0009) and a significant increase in new epithelial area (P = 0.007), but had no effect on reepithelialization. In deep partial thickness burns, rKGF induced the most consistent changes. Daily application of rKGF induced a highly significant increase in new epithelial area (P < 0.0001) but induced only a modest increase in reepithelialization (83.7% rKGF-treated versus 70.2% control; P = 0.016) 12 days postburn. rKGF also doubled the number of fully reepithelialized burns (P = 0.02) at 13 days postburn, at least partially because of marked stimulation of both epidermal and follicular proliferation as assessed by proliferating cell nuclear antigen expression. In situ hybridization for

  12. Angiopoietin-related growth factor (AGF) supports adhesion, spreading, and migration of keratinocytes, fibroblasts, and endothelial cells through interaction with RGD-binding integrins

    SciTech Connect

    Zhang Yueqing; Hu Xiaobo; Tian Ruiyang; Wei Wangui; Hu Wei; Chen Xia; Han Wei; Chen Huayou; Gong Yi . E-mail: ygong@sibs.ac.cn

    2006-08-18

    Angiopoietin-related growth factor (AGF) is a newly identified member of angiopoietin-related proteins (ARPs)/angiopoietin-like proteins (Angptls). AGF has been considered as a novel growth factor in accelerating cutaneous wound healing, as it is capable of stimulating keratinocytes proliferation as well as angiogenesis. But in our paper, we demonstrate that AGF stimulates keratinocytes proliferation only at high protein concentration, however, it can potently promote adhesion, spreading, and migration of keratinocytes, fibroblasts, and endothelial cells. Furthermore, we confirm that the adhesion and migration cellular events are mediated by RGD-binding integrins, most possibly the {alpha}{sub v}-containing integrins, by in vitro inhibition assays using synthetic competitive peptides. Our results strongly suggest that AGF is an integrin ligand as well as a mitogenic growth factor and theoretically participates in cutaneous wound healing in a more complex mechanism.

  13. Photodynamic therapy inhibit Fibroblast Growth Factor-10 induced keratinocyte differentiation and proliferation through ROS in Fibroblast Growth Factor Receptor-2b pathway

    PubMed Central

    Gozali, Maya Valeska; Yi, Fei; Zhang, Jia-an; Liu, Juan; Wu, Hong-jin; Xu, Yang; Luo, Dan; Zhou, Bing-rong

    2016-01-01

    5-aminolevulinic acid-photodynamic therapy (ALA-PDT) is known to be effective in several skin diseases such as acne, actinic keratoses, condyloma acuminata. However, some detailed mechanisms of ALA-PDT to treat these skin diseases still remain elusive. In this study, we aimed to investigate mechanism of ALA-PDT in in-vitro and in-vivo models. For in vitro, we use human keratinocyte cell line (HaCaT) cells. CCK-8 was used to detect cell proliferation activity, immunofluorescence and western blotting method to detect the content of keratin (K)1, K6, K16, protein kinase C (PKC), fibroblast growth factor receptor-2b (FGFR2b) protein, ELISA and RT-PCR to detect expression of interleukin (IL) 1α in the cell supernatant, and detect reactive oxygen species (ROS). For in vivo, we use 20 rabbits to induce hyperkeratosis acne model in their ear. Dermatoscope was used to see follicle hyperkeratosis and skin biopsy to analyze histology and immunohistochemical of PKC, FGFR2b, K1, K6 and K16. Results from this study suggest that ROS stimulated by ALA-PDT lead to inhibition of FGFR2b pathway in PKC downstream to cause reduction of IL1α expression, and eventually, keratinocytes differentiation and proliferation. Our data thus reveal a treatment mechanism of ALA-PDT underlying hyperkeratosis related dermatoses. PMID:27273653

  14. Human skin model containing melanocytes: essential role of keratinocyte growth factor for constitutive pigmentation-functional response to α-melanocyte stimulating hormone and forskolin.

    PubMed

    Duval, Christine; Chagnoleau, Corinne; Pouradier, Florence; Sextius, Peggy; Condom, Elodie; Bernerd, Françoise

    2012-12-01

    To study human skin pigmentation in a physiological in vitro model, we developed a pigmented reconstructed skin reproducing the three-dimensional architecture of the melanocyte environment and the interactions of melanocyte with its cellular partners, keratinocytes, and fibroblasts. Co-seeding melanocytes and keratinocytes onto a fibroblast-populated collagen matrix led to a correct integration of melanocytes within the epidermal basal layer, but melanocytes remained amelanotic even after supplementation with promelanogenic factors. Interestingly, normalization of keratinocyte differentiation using keratinocyte growth factor instead of epidermal growth factor finally allowed an active pigmentary system to develop, as shown by the expression of key melanogenic markers, the production, and transfer of melanosome-containing melanin into keratinocytes. Various degrees of constitutive pigmentation were reproduced using melanocytes from different skin phenotypes. Furthermore, induction of pigmentation was achieved by treatment with known propigmenting molecules, αMSH and forskolin, thus demonstrating the functionality of the pigmentary system. This pigmented full-thickness skin model therefore represents a highly relevant tool to study the role of cell-cell, cell-matrix, and mesenchymal-epithelial interactions in the control of skin pigmentation.

  15. Differential cell cycle response of nontumorigenic and tumorigenic human papillomavirus-positive keratinocytes towards transforming growth factor-beta1.

    PubMed

    Hasskarl, J; Butz, K; Whitaker, N; Ullmann, A; Dürst, M; Hoppe-Seyler, F

    2000-01-01

    Human papillomaviruses (HPVs) are causative agents of a number of malignancies in humans, including cervical cancer. Their tumorigenic potential is linked to expression of the viral E6/E7 genes which can interfere with normal cell cycle control by targeting p53, p21WAF1, p27KIP1, and pRb. We show here that nontumorigenic and tumorigenic HPV-positive keratinocytes (HPK) exhibit striking differences in the response of cell cycle regulatory genes towards transforming growth factor beta-beta1. Treatment with this agent led to an efficient induction of p53 and the growth-inhibitory p15INK4 and p21WAF1 genes only in nontumorigenic HPKs and was linked to an efficient reduction in viral E6/E7 oncogene expression. This was associated with increased pRb levels, exhibiting sustained hypophosphorylation, and a permanent growth arrest in the G1 phase of the cell cycle. In contrast, tumorigenic HPKs exhibited only a modest rise in p53 protein levels and a substantially reduced induction of the p15INK4 and p21WAF1 genes, which was linked to a lesser degree of viral oncogene repression. In addition, tumorigenic HPKs rapidly resumed cell growth after a transient G1 arrest, concomitantly with the reappearance of hyperphosphorylated pRb. These results support the notion that the progression of HPV-positive cells to a malignant phenotype is associated with increased resistance to growth inhibition by transforming growth factor-beta1. This is linked in the tumorigenic cells to a lack of persistent G1 arrest, inefficient induction of several cell cycle control genes involved in growth inhibition, and inefficient repression of the growth-promoting viral E6/E7 oncogenes. PMID:10794545

  16. Interactions between stromal cell--derived keratinocyte growth factor and epithelial transforming growth factor in immune-mediated crypt cell hyperplasia.

    PubMed Central

    Bajaj-Elliott, M; Poulsom, R; Pender, S L; Wathen, N C; MacDonald, T T

    1998-01-01

    Immune reactions in the gut are associated with increased epithelial cell proliferation. Here we have studied the role of keratinocyte growth factor (KGF; FGF7) and transforming growth factor-alpha (TGF-alpha) in the epithelial cell hyperplasia seen in explants of fetal human small intestine after activation of lamina propria T cells with the superantigen Staphylococcus aureus enterotoxin B (SEB). After the addition of SEB to the explants there is a 10-fold increase in KGF mRNA by 72 h of culture. KGF transcripts were abundant in the lamina propria using in situ hybridization and the culture supernatants contained elevated amounts of KGF protein. SEB had no direct effect on KGF mRNA and protein production by cultured lamina propria mesenchymal cells, but both were upregulated by TNF-alpha. Accompanying the increase in KGF there was also an increase in TGF-alpha precursor proteins in the culture supernatants and the phosphorylated form of the EGFR receptor was also detected in the tissue. Increased TGF-alpha precursor proteins were also detected in the supernatants of control explants stimulated with KGF alone. The direct addition of KGF and TGF-alpha enhanced epithelial cell proliferation and antibodies against KGF and TGF-alpha partially inhibited SEB-induced crypt hyperplasia. These results suggest molecular cross-talk between the KGF/KGFR and the TGF-alpha/EGFR in immune-mediated crypt cell hyperplasia. PMID:9788959

  17. Acemannan stimulates gingival fibroblast proliferation; expressions of keratinocyte growth factor-1, vascular endothelial growth factor, and type I collagen; and wound healing.

    PubMed

    Jettanacheawchankit, Suwimon; Sasithanasate, Siriruk; Sangvanich, Polkit; Banlunara, Wijit; Thunyakitpisal, Pasutha

    2009-04-01

    Aloe vera has long been used as a traditional medicine for inducing wound healing. Gingival fibroblasts (GFs) play an important role in oral wound healing. In this study, we investigated the effects of acemannan, a polysaccharide extracted from Aloe vera gel, on GF proliferation; keratinocyte growth factor-1 (KGF-1), vascular endothelial growth factor (VEGF), and type I collagen production; and oral wound healing in rats. [(3)H]-Thymidine incorporation assay and ELISA were used. Punch biopsy wounds were created at the hard palate of male Sprague Dawley rats. All treatments (normal saline; 0.1% triamcinolone acetonide; plain 1% Carbopol; and Carbopol containing 0.5%, 1%, and 2% acemannan (w/w)) were applied daily. Wounded areas and histological features were observed at day 7 after treatment. From our studies, acemannan at concentrations of 2, 4, 8, and 16 mg/ml significantly induced cell proliferation (P<0.05). Acemannan concentrations between 2 - 16 mg/ml significantly stimulated KGF-1, VEGF, and type I collagen expressions (P<0.05). Wound healing of animals receiving Carbopol containing 0.5% acemannan (w/w) was significantly better than that of the other groups (P<0.05). These findings suggest that acemannan plays a significant role in the oral wound healing process via the induction of fibroblast proliferation and stimulation of KGF-1, VEGF, and type I collagen expressions.

  18. A protective role for keratinocyte growth factor in a murine model of chemotherapy and radiotherapy-induced mucositis

    SciTech Connect

    Borges, Luis . E-mail: borgesl@amgen.com; Rex, Karen L.; Chen, Jennifer N.; Wei, Ping; Kaufman, Stephen; Scully, Sheila; Pretorius, James K.; Farrell, Catherine L.

    2006-09-01

    Purpose: To evaluate the activity of palifermin (rHuKGF) in a murine model of mucosal damage induced by a radiotherapy/chemotherapy (RT/CT) regimen mimicking treatment protocols used in head-and-neck cancer patients. Methods and Materials: A model of mucosal damage induced by RT/CT was established by injecting female BDF1 mice with cisplatin (10 mg/kg) on Day 1; 5-fluorouracil (40 mg/kg/day) on Days 1-4, and irradiation (5 Gy/day) to the head and neck on Days 1-5. Palifermin was administered subcutaneously on Days -2 to 0 (5 mg/kg/day) and on Day 5 (5 mg/kg). Evaluations included body weight, organ weight, keratinocyte growth factor receptor expression, epithelial thickness, and cellular proliferation. Results: Initiation of the radiochemotherapeutic regimen resulted in a reduction in body weight in control animals. Palifermin administration suppressed weight loss and resulted in increased organ weight (salivary glands and small intestine), epithelial thickness (esophagus and tongue), and cellular proliferation (tongue and salivary glands). Conclusions: Administration of palifermin before RT/CT promotes cell proliferation and increases in epithelial thickness in the oral mucosa, salivary glands, and digestive tract. Palifermin administration before and after RT/CT mitigates weight loss and a trophic effect on the intestinal mucosa and salivary glands, suggesting that palifermin use should be investigated further in the RT/CT settings, in which intestinal mucositis and salivary gland dysfunction are predominant side effects of cytotoxic therapy.

  19. Regulation of transglutaminase type II by transforming growth factor-beta 1 in normal and transformed human epidermal keratinocytes.

    PubMed

    George, M D; Vollberg, T M; Floyd, E E; Stein, J P; Jetten, A M

    1990-07-01

    This study examines the effect of transforming growth factor-beta 1 (TGF-beta 1) on the expression of Type I and II transglutaminase in normal human epidermal keratinocytes (NHEK cells). Treatment of undifferentiated NHEK cells with 100 pM TGF-beta 1 caused a 10- to 15-fold increase in the activity of a soluble transglutaminase. Based on its cellular distribution and immunoreactivity this transglutaminase was identified as Type II (tissue) transglutaminase. TGF-beta 1 did not enhance the levels of the membrane-bound Type I (epidermal) transglutaminase activity which is induced during squamous cell differentiation and did not increase Type II transglutaminase activity in differentiated NHEK cells. Several SV40 large T antigen-immortalized NHEK cell lines also exhibited a dramatic increase in transglutaminase Type II activity after TGF-beta 1 treatment; however, TGF-beta 1 did not induce any significant change in transglutaminase activity in the carcinoma-derived cell lines SCC-13, SCC-15, and SQCC/Y1. Half-maximal stimulation of transglutaminase Type II activity in NHEK cells occurred at a dose of 15 pM TGF-beta 1. TGF-beta 2 was about equally effective. This enhancement in transglutaminase activity was related to an increase in the amount of transglutaminase Type II protein as indicated by immunoblot analysis. Northern blot analyses using a specific cDNA probe for Type II transglutaminase showed that exposure of NHEK cells to TGF-beta 1 caused a marked increase in the mRNA levels of this enzyme which could be observed as early as 4 h after the addition of TGF-beta 1. Maximal induction of transglutaminase Type II mRNA occurred between 18 and 24 h. The increase in Type II transglutaminase mRNA levels was blocked by the presence of cycloheximide, suggesting that this increase in mRNA by TGF-beta 1 is dependent on protein synthesis. PMID:1972706

  20. Reduction of radiochemotherapy-induced early oral mucositis by recombinant human keratinocyte growth factor (palifermin): Experimental studies in mice

    SciTech Connect

    Doerr, Wolfgang . E-mail: doerr@rcs.urz.tu-dresden.de; Baessler, Stefan; Reichel, Sandra; Spekl, Kathrin

    2005-07-01

    Purpose: To study the effect of recombinant human keratinocyte growth factor (rHuKGF or palifermin) on oral mucositis induced by radiochemotherapy in a mouse model. Methods and Materials: Cis-diamminedichloroplatinum (cisplatin) and/or 5-fluorouracil were given before single dose irradiation, combined with palifermin before or after the treatment, or both. Daily fractionated irradiation for 2 weeks was followed by graded test doses. With additional chemotherapy in Week 1, palifermin was given before radiotherapy and at the end of the first week, or additionally at the end of Week 2. Radiochemotherapy in Week 2 was combined with palifermin at the end of Weeks 1 and 2, Weeks 1, 2, and 3, or additionally before radiotherapy. Ulceration of mouse tongue mucosa was analyzed as the endpoint. Results: The dose associated with ulcer induction in 50% of the mice (ED{sub 50}) for single-dose irradiation was 11.5 {+-} 0.7 Gy. Palifermin increased the ED{sub 50} to about 19 Gy in all protocols tested. Similar values were observed when chemotherapy was added before irradiation. With fractionated irradiation, palifermin increased the ED{sub 50} for test irradiation from 5.7 {+-} 1.5 Gy to 12-15 Gy, depending on the administration protocol. With chemotherapy in Week 1, two palifermin injections had no significant effect, but a third injection increased the ED{sub 50} to 13 Gy. With chemotherapy in Week 2, all palifermin protocols resulted in ED{sub 50} values of 13-14 Gy. Conclusion: A marked increase in oral mucosal radiation tolerance by palifermin was found, which was preserved in combinations with chemotherapy using cisplatin and/or 5-fluorouracil.

  1. Regulation of growth and gene expression in human papillomavirus-transformed keratinocytes by transforming growth factor-beta: implications for the control of papillomavirus infection.

    PubMed

    Braun, L; Dürst, M; Mikumo, R; Crowley, A; Robinson, M

    1992-01-01

    Cervical carcinogenesis is a multistep process that appears to be initiated by infection of squamous epithelial cells in the cervix with one of a limited number of human papillomavirus (HPV) types. However, the mechanisms involved in the evolution of benign, HPV-induced lesions to malignancy have not yet been fully elucidated. Transforming growth factor-beta (TGF-beta), a multifunctional growth factor produced by cells in the skin, inhibits the proliferation of foreskin and cervical keratinocytes in vitro. We examined the effects of TGF-beta on growth and virus early-gene expression in cell lines immortalized by two HPV types associated with cervical carcinogenesis as well as the expression of TGF-beta 1 mRNA transcripts in normal and HPV-positive cells in vivo and in vitro. We found that normal and HPV-positive cells expressed similar levels of TGF-beta 1 mRNAs and exhibited similar patterns of responsiveness to three isoforms of TGF-beta in both monolayer and modified organotypic cultures. Of particular interest is our finding that the expression of the E6 and E7 early viral transforming regions of both HPV16 and HPV18 was reversibly and rapidly inhibited by TGF-beta. In one HPV16-positive cell line examined in detail, inhibition of HPV expression required protein synthesis and occurred at the level of transcription. HPV-immortalized cells selected for resistance to in vitro differentiation signals remained sensitive to TGF-beta-mediated growth inhibition. These results, showing that both growth and virus gene expression in HPV-transformed cells were responsive to TGF-beta, suggest that endogenous growth factors produced by different cell types in squamous epithelium may play a role in the progression of cervical neoplasia. PMID:1326988

  2. The protective effect of recombinant human keratinocyte growth factor on radiation-induced pulmonary toxicity in rats

    SciTech Connect

    Chen Liguang; Brizel, David M.; Rabbani, Zahid N.; Samulski, Thaddeus V.; Farrell, Catherine L.; Larrier, Nicole; Anscher, Mitchell S.; Vujaskovic, Zeljko . E-mail: vujas@radonc.duke.edu

    2004-12-01

    Purpose: Radiation-induced lung toxicity is a significant dose-limiting side effect of radiotherapy for thoracic tumors. Recombinant human keratinocyte growth factor (rHuKGF) has been shown to be a mitogen for type II pneumocytes. The purpose of this study was to determine whether rHuKGF prevents or ameliorates the severity of late lung damage from fractionated irradiation in a rat model. Methods and materials: Female Fisher 344 rats were irradiated to the right hemithorax with a dose of 40 Gy/5 fractions/5 days. rHuKGF at dose of 5 mg/kg or 15 mg/kg was given via a single intravenous injection 10 min after the last fraction of irradiation. Animals were followed for 6 months after irradiation. Results: The breathing rate increased beginning at 6 weeks and reached a peak at 14 weeks after irradiation. The average breathing frequencies in the irradiated groups with rHuKGF (5 mg/kg and 15 mg/kg) treatment were significantly lower than that in the group receiving radiation without rHuKGF (116.5 {+-} 1.0 and 115.2 {+-} 0.8 vs 123.5 {+-} 1.2 breaths/min, p < 0.01). The severity of lung fibrosis and the level of immunoreactivity of integrin {alpha}v{beta}6, TGF{beta}1, type II TGF{beta} receptor, Smad3, and phosphorylated Smad2/3 were significantly decreased only in the group receiving irradiation plus high-dose rHuKGF treatment compared with irradiation plus vehicle group, suggesting a dose response for the effect of rHuKGF. Conclusions: This study is the first to demonstrate that rHuKGF treatment immediately after irradiation protects against late radiation-induced pulmonary toxicity. These results suggest that restoration of the integrity of the pulmonary epithelium via rHuKGF stimulation may downregulate the TGF-{beta}-mediated fibrosis pathway. These data also support the use of rHuKGF in a clinical trial designed to prevent radiation-induced lung injury.

  3. Activation of the human keratinocyte B1 bradykinin receptor induces expression and secretion of metalloproteases 2 and 9 by transactivation of epidermal growth factor receptor.

    PubMed

    Matus, Carola E; Ehrenfeld, Pamela; Pavicic, Francisca; González, Carlos B; Concha, Miguel; Bhoola, Kanti D; Burgos, Rafael A; Figueroa, Carlos D

    2016-09-01

    The B1 bradykinin receptor (BDKRB1) is a component of the kinin cascade localized in the human skin. Some of the effects produced by stimulation of BDKRB1 depend on transactivation of epidermal growth factor receptor (EGFR), but the mechanisms involved in this process have not been clarified yet. The primary purpose of this study was to determine the effect of a BDKRB1 agonist on wound healing in a mouse model and the migration and secretion of metalloproteases 2 and 9 from human HaCaT keratinocytes and delineate the signalling pathways that triggered their secretion. Although stimulation of BDKRB1 induces weak chemotactic migration of keratinocytes and wound closure in an in vitro scratch-wound assay, the BDKRB1 agonist improved wound closure in a mouse model. BDKRB1 stimulation triggers synthesis and secretion of both metalloproteases, effects that depend on the activity of EGFR and subsequent phosphorylation of ERK1/2 and p38 mitogen-activated protein kinases and PI3K/Akt. In the mouse model, immunoreactivity for both gelatinases was concentrated around wound borders. EGFR transactivation by BDKRB1 agonist involves Src kinases family and ADAM17. In addition to extracellular matrix degradation, metalloproteases 2 and 9 regulate cell migration and differentiation, cell functions that are associated with the role of BDKRB1 in keratinocyte differentiation. Considering that BDKRB1 is up-regulated by inflammation and/or by cytokines that are abundant in the inflammatory milieu, more stable BDKRB1 agonists may be of therapeutic value to modulate wound healing.

  4. Epidermal Growth Factor Receptor Transactivation Is Required for Mitogen-Activated Protein Kinase Activation by Muscarinic Acetylcholine Receptors in HaCaT Keratinocytes

    PubMed Central

    Ockenga, Wymke; Kühne, Sina; Bocksberger, Simone; Banning, Antje; Tikkanen, Ritva

    2014-01-01

    Non-neuronal acetylcholine plays a substantial role in the human skin by influencing adhesion, migration, proliferation and differentiation of keratinocytes. These processes are regulated by the Mitogen-Activated Protein (MAP) kinase cascade. Here we show that in HaCaT keratinocytes all five muscarinic receptor subtypes are expressed, but M1 and M3 are the subtypes involved in mitogenic signaling. Stimulation with the cholinergic agonist carbachol leads to activation of the MAP kinase extracellular signal regulated kinase, together with the protein kinase Akt. The activation is fully dependent on the transactivation of the epidermal growth factor receptor (EGFR), which even appears to be the sole pathway for the muscarinic receptors to facilitate MAP kinase activation in HaCaT cells. The transactivation pathway involves a triple-membrane-passing process, based on activation of matrix metalloproteases, and extracellular ligand release; whereas phosphatidylinositol 3-kinase, Src family kinases or protein kinase C do not appear to be involved in MAP kinase activation. Furthermore, phosphorylation, ubiquitination and endocytosis of the EGF receptor after cholinergic transactivation are different from that induced by a direct stimulation with EGF, suggesting that ligands other than EGF itself mediate the cholinergic transactivation. PMID:25421240

  5. Activated Protein C Enhances Human Keratinocyte Barrier Integrity via Sequential Activation of Epidermal Growth Factor Receptor and Tie2*

    PubMed Central

    Xue, Meilang; Chow, Shu-Oi; Dervish, Suat; Chan, Yee-Ka Agnes; Julovi, Sohel M.; Jackson, Christopher J.

    2011-01-01

    Keratinocytes play a critical role in maintaining epidermal barrier function. Activated protein C (APC), a natural anticoagulant with anti-inflammatory and endothelial barrier protective properties, significantly increased the barrier impedance of keratinocyte monolayers, measured by electric cell substrate impedance sensing and FITC-dextran flux. In response to APC, Tie2, a tyrosine kinase receptor, was rapidly activated within 30 min, and relocated to cell-cell contacts. APC also increased junction proteins zona occludens, claudin-1 and VE-cadherin. Inhibition of Tie2 by its peptide inhibitor or small interfering RNA abolished the barrier protective effect of APC. Interestingly, APC did not activate Tie2 through its major ligand, angiopoietin-1, but instead acted by binding to endothelial protein C receptor, cleaving protease-activated receptor-1 and transactivating EGF receptor. Furthermore, when activation of Akt, but not ERK, was inhibited, the barrier protective effect of APC on keratinocytes was abolished. Thus, APC activates Tie2, via a mechanism requiring, in sequential order, the receptors, endothelial protein C receptor, protease-activated receptor-1, and EGF receptor, which selectively enhances the PI3K/Akt signaling to enhance junctional complexes and reduce keratinocyte permeability. PMID:21173154

  6. A novel signaling pathway of tissue kallikrein in promoting keratinocyte migration: Activation of proteinase-activated receptor 1 and epidermal growth factor receptor

    SciTech Connect

    Gao, Lin; Chao, Lee; Chao, Julie

    2010-02-01

    Biological functions of tissue kallikrein (TK, KLK1) are mainly mediated by kinin generation and subsequent kinin B2 receptor activation. In this study, we investigated the potential role of TK and its signaling pathways in cultured human keratinocyte migration and in a rat skin wound healing model. Herein, we show that TK promoted cell migration and proliferation in a concentration- and time-dependent manner. Inactive TK or kinin had no significant effect on cell migration. Interestingly, cell migration induced by active TK was not blocked by icatibant or L-NAME, indicating an event independent of kinin B2 receptor and nitric oxide formation. TK's stimulatory effect on cell migration was inhibited by small interfering RNA for proteinase-activated receptor 1 (PAR{sub 1}), and by PAR{sub 1} inhibitor. TK-induced migration was associated with increased phosphorylation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK), which was blocked by inhibition of protein kinase C (PKC), Src, EGFR and ERK. TK-induced cell migration and EGFR phosphorylation were blocked by metalloproteinase (MMP) inhibitor, heparin, and antibodies against EGFR external domain, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin (AR). Local application of TK promoted skin wound healing in rats, whereas icatibant and EGFR inhibitor blocked TK's effect. Skin wound healing was further delayed by aprotinin and neutralizing TK antibody. This study demonstrates a novel role of TK in skin wound healing and uncovers new signaling pathways mediated by TK in promoting keratinocyte migration through activation of the PAR{sub 1}-PKC-Src-MMP pathway and HB-EGF/AR shedding-dependent EGFR transactivation.

  7. The E5 oncoprotein of human papillomavirus type 16 enhances endothelin-1-induced keratinocyte growth.

    PubMed

    Venuti, A; Salani, D; Poggiali, F; Manni, V; Bagnato, A

    1998-08-15

    Human keratinocytes express ETA receptors and produce endothelin-1 (ET-1), which stimulates growth response. Previously, we reported that a twofold increase in ETA receptors is present in human papillomavirus type 16 (HPV16) immortalized keratinocytes and that ET-1 induces enhanced proliferative response in these cell lines compared to normal cells. The present studies examine whether the E5 gene of HPV16 is responsible for the enhanced activity of ET-1 in HPV-transfected keratinocytes. The presence of the E5 gene in growth factor-starved keratinocytes induced the DNA synthesis and enhanced the mitogenic activity of ET-1 or epidermal growth factor. The selection of primary keratinocytes in growth factor-free medium with the addition of ET-1 as a growth factor showed that E5-transfected keratinocytes were able to grow and to form a higher number of larger colonies with respect to untransfected cells. This effect seems to be related to the interaction of E5 with the mitogenic signaling pathway of ET-1 rather than to an increase in the expression of the receptors for ET-1. In conclusion, our data demonstrate that E5 enhances ligand signaling in keratinocytes outside the EGF pathway by the amplification of the proliferative effect of ET-1/ETA receptor signaling.

  8. Role of Keratinocyte Growth Factor in the Differentiation of Sweat Gland-Like Cells From Human Umbilical Cord-Derived Mesenchymal Stem Cells

    PubMed Central

    Xu, Yongan; Hong, Yucai; Xu, Mengyan; Ma, Kui; Fu, Xiaobing

    2016-01-01

    Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have higher proliferation potency and lower immune resistance than human bone marrow MSCs and can differentiate into various functional cells. Many regulatory factors, including keratinocyte growth factor (KGF), are involved in the development of skin and cutaneous appendages. Although KGF is important in wound healing, the role of KGF in hUC-MSC differentiation remains unknown. In our previous work, we found the mixing medium (nine parts of basic sweat-gland [SG] medium plus one part of conditioned heat-shock SG medium) could induce hUC-MSC differentiation to sweat gland-like cells (SGCs). In this study, we further improved the inducing medium and determined the effects of KGF in hUC-MSC differentiation. We found KGF expression in the SGCs and that recombinant human KGF could induce hUC-MSC differentiation into SGCs, suggesting KGF plays a pivotal role in promoting hUC-MSC differentiation to SGCs. Furthermore, the SGCs differentiated from hUC-MSCs were applied to severely burned skin of the paw of an in vivo severe combined immunodeficiency mouse burn model. Burned paws treated with SGCs could regenerate functional sparse SGs 21 days after treatment; the untreated control paws could not. Collectively, these results demonstrated that KGF is a critical growth factor for SGC differentiation from hUC-MSCs and the differentiated SGCs from hUC-MSCs may have a potential therapeutic application for regeneration of destroyed SGs and injured skin. Significance There is growing evidence demonstrating a potential therapeutic application of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) in injured skin. In the current study, conditioned media and chemically defined media with recombinant human keratinocyte growth factor (KGF) could induce hUC-MSC differentiation into sweat gland-like cells (SGCs). Moreover, the differentiated SGCs from hUC-MSCs could regenerate functional sparse sweat glands in a

  9. Storage stability of keratinocyte growth factor-2 in lyophilized formulations: effects of formulation physical properties and protein fraction at the solid-air interface.

    PubMed

    Devineni, Dilip; Gonschorek, Christoph; Cicerone, Marcus T; Xu, Yemin; Carpenter, John F; Randolph, Theodore W

    2014-10-01

    Lyophilized formulations of keratinocyte growth factor-2 (KGF-2) were prepared with a range of disaccharide (sucrose or trehalose) and hydroxyethyl starch (HES) mass ratios. Protein degradation was assessed as a function of time of storage of the dried formulations at 40, 50 and 60°C. Lyophilized and stored samples were rehydrated, and protein degradation was quantified by measuring loss of monomeric protein with size exclusion chromatography and by determining chemical degradation in the soluble fraction with reverse-phase chromatography. The secondary structure of the protein in the lyophilized formulations was studied with infrared spectroscopy. The magnitudes of degradation were compared the key physical properties of the formulations including retention of protein native secondary structure, glass transition temperature (Tg), inverse mean square displacements 〈u(2)〉(-1) for hydrogen atoms (fast β relaxation), and the relaxation time τ(β), which correlates with relaxation due to fast Johari-Goldstein motions in the glass (Xu et al., 2013) [1]. In addition, specific surface areas of the lyophilized formulations were determined by Brunauer-Emmet-Teller analysis of krypton adsorption isotherms and used to estimate the fraction of the KGF-2 molecules residing at the solid-air interface. KGF-2 degradation rates were highest in formulations wherein the protein's structure was most perturbed, and wherein β relaxations were fastest, but the dominant factor governing KGF-2 degradation in freeze-dried formulations was the fraction of the protein found at the glass solid-air interface. PMID:24859390

  10. Epidermal Growth Factor Receptor Is a Critical Mediator of Ultraviolet B Irradiation-Induced Signal Transduction in Immortalized Human Keratinocyte HaCaT Cells

    PubMed Central

    Xu, Yiru; Voorhees, John J.; Fisher, Gary J.

    2006-01-01

    Epidermal growth factor receptor (EGFR) is a critical mediator of several types of epithelial cancers. Skin cancer arising from exposure to ultraviolet B irradiation (UVB) from the sun is a prominent form of human cancer. Recent data indicate that in addition to cognate ligands, EGFR is activated by UVB irradiation. We used pharmacological and genetic approaches to investigate the function of EGFR in mediating UVB-induced signal transduction in human skin keratinocyte HaCaT cells. Pharmacological inhibition of EGFR tyrosine kinase significantly inhibited UVB-mediated induction of ERK, p38, and JNK MAP kinases, and their effectors, transcription factors c-Fos and c-Jun. Inhibition of UVB activation of EGFR also suppressed activation of AKT-, PKC-, and PKA-dependent signal transduction pathways. B82 mouse L cells devoid of EGFR were used to further investigate EGFR dependence of UVB-induced signal transduction. UVB failed to induce ERK, and JNK activation was reduced 60% in B82 cells compared to B82K+ cells, which express EGFR. In addition, UVB induced both c-Fos and c-Jun proteins in B82K+ cells, whereas neither were induced in B82 cells. Taken together, these data demonstrate that EGFR is required for UVB-mediated induction of multiple signaling pathways that are known to mediate tumor formation in skin. PMID:16936259

  11. The Inflammasome and the Epidermal Growth Factor Receptor (EGFR) Are Involved in the Staphylococcus aureus-Mediated Induction of IL-1alpha and IL-1beta in Human Keratinocytes

    PubMed Central

    Schröder, Lena; Gläser, Regine; Harder, Jürgen

    2016-01-01

    Staphylococcus (S.) aureus is an important pathogen causing various infections including those of the skin. Keratinocytes are able to sense invading S. aureus and to initiate a fast defense reaction by the rapid release of innate defense mediators such as antimicrobial peptides and cytokines. There is increasing evidence that the cytokines IL-1alpha and IL-1beta, which both signal through the IL-1 receptor, play an important role in cutaneous defense against S. aureus. The aim of this study was to gain more insight into the underlying mechanisms leading to the S. aureus-induced IL-1alpha and IL-1beta expression in keratinocytes. Infection of human primary keratinocytes with S. aureus led to the induction of gene expression and protein secretion of IL-1alpha and IL-1beta. Full S. aureus-induced IL-1 protein release required the inflammasome components caspase-1 and ASC (apoptosis-associated speck-like protein containing a CARD) whereas gene induction of IL-1alpha and IL-beta by S. aureus was not dependent on caspase-1 and ASC. Since patients receiving anti-cancer therapy by inhibition of the epidermal growth factor receptor (EGFR) often suffer from skin infections caused by S. aureus we additionally evaluated whether the EGFR pathway may be involved in the IL-1alpha and IL-1beta induction by S. aureus. Inactivation of the EGFR with a blocking antibody decreased the S. aureus-mediated IL-1alpha and IL-1beta induction in primary keratinocytes. Moreover, the use of siRNA experiments revealed that ADAM17 (A Disintegrin and A Metalloprotease 17), a metalloproteinase known to mediate the shedding and release of EGFR ligands, was required for full induction of IL-1alpha and IL-1beta in keratinocytes infected with S. aureus. A failure of keratinocytes to adequately upregulate IL-1alpha and IL-1beta may promote S. aureus skin infections. PMID:26808616

  12. Keratinocyte Growth Factor Gene Electroporation into Skeletal Muscle as a Novel Gene Therapeutic Approach for Elastase-Induced Pulmonary Emphysema in Mice

    PubMed Central

    Tobinaga, Shuichi; Matsumoto, Keitaro; Nagayasu, Takeshi; Furukawa, Katsuro; Abo, Takafumi; Yamasaki, Naoya; Tsuchiya, Tomoshi; Miyazaki, Takuro; Koji, Takehiko

    2015-01-01

    Pulmonary emphysema is a progressive disease with airspace destruction and an effective therapy is needed. Keratinocyte growth factor (KGF) promotes pulmonary epithelial proliferation and has the potential to induce lung regeneration. The aim of this study was to determine the possibility of using KGF gene therapy for treatment of a mouse emphysema model induced by porcine pancreatic elastase (PPE). Eight-week-old BALB/c male mice treated with intra-tracheal PPE administration were transfected with 80 μg of a recombinant human KGF (rhKGF)-expressing FLAG-CMV14 plasmid (pKGF-FLAG gene), or with the pFLAG gene expressing plasmid as a control, into the quadriceps muscle by electroporation. In the lung, the expression of proliferating cell nuclear antigen (PCNA) was augmented, and surfactant protein A (SP-A) and KGF receptor (KGFR) were co-expressed in PCNA-positive cells. Moreover, endogenous KGF and KGFR gene expression increased significantly by pKGF-FLAG gene transfection. Arterial blood gas analysis revealed that the PaO2 level was not significantly reduced on day 14 after PPE instillation with pKGF-FLAG gene transfection compared to that of normal mice. These results indicated that KGF gene therapy with electroporation stimulated lung epithelial proliferation and protected depression of pulmonary function in a mouse emphysema model, suggesting a possible method of treating pulmonary emphysema. PMID:26160987

  13. Keratinocyte growth factor in inflammatory bowel disease. Increased mRNA transcripts in ulcerative colitis compared with Crohn's disease in biopsies and isolated mucosal myofibroblasts.

    PubMed Central

    Bajaj-Elliott, M.; Breese, E.; Poulsom, R.; Fairclough, P. D.; MacDonald, T. T.

    1997-01-01

    Inflammation in the gastrointestinal tract is associated with increased epithelial cell proliferation. Keratinocyte growth factor (KGF) is an epithelial cell mitogen widely expressed by mesenchymal cells subjacent to the epithelial cells. In this study, we have investigated the expression and distribution of KGF in normal and diseased (Crohn's disease and ulcerative colitis(UC)) intestine by quantitative competitive reverse-transcriptase polymerase chain reaction in whole biopsies and purified lamina propria myofibroblasts and by in situ hybridization. Analysis of whole mucosal biopsies reveals significantly higher numbers of KGF mRNA transcripts in UC compared with Crohn's colitis and control colon (P < 0.001). KGF transcripts were also elevated in Crohn's ileitis compared with normal ileum. In situ hybridization showed a marked increase in cells expressing KGF mRNA throughout the lamina propria in both UC and Crohn's tissue. In Crohn's disease, positively hybridizing cells were only rarely seen in the submucosa but were abundant around the bases of the crypts and were not associated with lymphoid aggregates. In purified mucosal myofibroblasts, increased (15- to 20-fold) KGF mRNA expression was seen in UC compared with control and Crohn's tissue. These results confirm and extend earlier studies showing that KGF transcripts are elevated in inflammatory bowel disease, but they show for the first time that transcripts are higher in UC than Crohn's disease because of increased production by mucosal mesenchymal cells. Images Figure 2 Figure 3 PMID:9358773

  14. Keratinocyte Growth Factor Gene Electroporation into Skeletal Muscle as a Novel Gene Therapeutic Approach for Elastase-Induced Pulmonary Emphysema in Mice.

    PubMed

    Tobinaga, Shuichi; Matsumoto, Keitaro; Nagayasu, Takeshi; Furukawa, Katsuro; Abo, Takafumi; Yamasaki, Naoya; Tsuchiya, Tomoshi; Miyazaki, Takuro; Koji, Takehiko

    2015-06-29

    Pulmonary emphysema is a progressive disease with airspace destruction and an effective therapy is needed. Keratinocyte growth factor (KGF) promotes pulmonary epithelial proliferation and has the potential to induce lung regeneration. The aim of this study was to determine the possibility of using KGF gene therapy for treatment of a mouse emphysema model induced by porcine pancreatic elastase (PPE). Eight-week-old BALB/c male mice treated with intra-tracheal PPE administration were transfected with 80 μg of a recombinant human KGF (rhKGF)-expressing FLAG-CMV14 plasmid (pKGF-FLAG gene), or with the pFLAG gene expressing plasmid as a control, into the quadriceps muscle by electroporation. In the lung, the expression of proliferating cell nuclear antigen (PCNA) was augmented, and surfactant protein A (SP-A) and KGF receptor (KGFR) were co-expressed in PCNA-positive cells. Moreover, endogenous KGF and KGFR gene expression increased significantly by pKGF-FLAG gene transfection. Arterial blood gas analysis revealed that the PaO2 level was not significantly reduced on day 14 after PPE instillation with pKGF-FLAG gene transfection compared to that of normal mice. These results indicated that KGF gene therapy with electroporation stimulated lung epithelial proliferation and protected depression of pulmonary function in a mouse emphysema model, suggesting a possible method of treating pulmonary emphysema.

  15. Delivery of plasmid DNA expression vector for keratinocyte growth factor-1 using electroporation to improve cutaneous wound healing in a septic rat model.

    PubMed

    Lin, Michael P; Marti, Guy P; Dieb, Rami; Wang, Jiaai; Ferguson, Mark; Qaiser, Rabia; Bonde, Pramod; Duncan, Mark D; Harmon, John W

    2006-01-01

    We have previously shown that wound healing was improved in a diabetic mouse model of impaired wound healing following transfection with keratinocyte growth factor-1 (KGF-1) cDNA. We now extend these findings to the characterization of the effects of DNA plasmid vectors delivered to rats using electroporation (EP) in vivo in a sepsis-based model of impaired wound healing. To assess plasmid transfection and wound healing, gWIZ luciferase and PCDNA3.1/KGF-1 expression vectors were used, respectively. Cutaneous wounds were produced using an 8 mm-punch biopsy in Sprague-Dawley rats in which healing was impaired by cecal ligation-induced sepsis. We used National Institutes of Health image analysis software and histologic assessment to analyze wound closure and found that EP increased expression of gWIZ luciferase vector up to 53-fold compared with transfection without EP (p < 0.001). EP-assisted plasmid transfection was found to be localized to skin. Septic rats had a 4.7 times larger average wound area on day 9 compared with control (p < 0.001). Rats that underwent PCDNA3.1/KGF-1 transfection with EP had 60% smaller wounds on day 12 compared with vector without EP (p < 0.009). Quality of healing with KGF-1 vector plus EP scored 3.0 +/- 0.3 and was significantly better than that of 1.8 +/- 0.3 for treatment with vector alone (p < 0.05). We conclude that both the rate and quality of healing were improved with DNA plasmid expression vector for growth factor delivered with EP to septic rats.

  16. AMPK regulation of the growth of cultured human keratinocytes

    SciTech Connect

    Saha, Asish K. . E-mail: aksaha@bu.edu; Persons, Kelly; Safer, Joshua D.; Luo Zhijun; Holick, Michael F.; Ruderman, Neil B.

    2006-10-20

    AMP kinase (AMPK) is a fuel sensing enzyme that responds to cellular energy depletion by increasing processes that generate ATP and inhibiting others that require ATP but are not acutely necessary for survival. In the present study, we examined the relationship between AMPK activation and the growth (proliferation) of cultured human keratinocytes and assessed whether the inhibition of keratinocyte growth by vitamin D involves AMPK activation. In addition, we explored whether the inhibition of keratinocyte proliferation as they approach confluence could be AMPK-related. Keratinocytes were incubated for 12 h with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-{beta}-D-ribofuranoside (AICAR). At concentrations of 10{sup -4} and 10{sup -3} M, AICAR inhibited keratinocyte growth by 50% and 95%, respectively, based on measurements of thymidine incorporation into DNA. It also increased AMPK and acetyl CoA carboxylase phosphorylation (P-AMPK and P-ACC) and decreased the concentration of malonyl CoA confirming that AMPK activation had occurred. Incubation with the thiazolidinedione, troglitazone (10{sup -6} M) caused similar alterations in P-AMPK, P-ACC, and cell growth. In contrast, the well known inhibition of keratinocyte growth by 1,25-dihydroxyvitamin D{sub 3} (10{sup -7} and 10{sup -6} M) was not associated with changes in P-AMPK or P-ACC. Like most cells, the growth of keratinocytes diminished as they approached confluence. Thus, it was of note that we found a progressive increase in P-AMPK (1.5- to 2-fold, p < 0.05) as keratinocytes grown in control medium went from 25% to 100% confluence. In conclusion, the data are consistent with the hypothesis that activation of AMPK acts as a signal to diminish the proliferation of cultured keratinocytes as they approach confluence. They also suggest that AMPK activators, such as AICAR and troglitazone, inhibit keratinocyte growth and that the inhibition of cell growth by 1,25-dihydroxyvitamin D{sub 3} is AMPK-independent.

  17. The autonomous growth of human papillomavirus type 16-immortalized keratinocytes is related to the endothelin-1 autocrine loop.

    PubMed

    Venuti, A; Marcante, M L; Flamini, S; Di Castro, V; Bagnato, A

    1997-09-01

    Some human papillomaviruses (HPVs) such as HPV type 16 (HPV16) and HPV18 are involved in cervical carcinoma, and they can immortalize and transform keratinocytes. Endothelin-1 (ET-1) is produced in keratinocytes and has been shown to act through ETA receptors as an autocrine growth factor for keratinocytes. This study examines whether HPV16 alters the ET-1-mediated autocrine loop in human keratinocytes, providing a selective growth advantage for transformed cells. ET-1 is released in similar amounts from normal and HPV-transfected keratinocytes. All HPV-transfected cell lines express high-affinity ETA receptors. A two-fold increase in ET-1 binding sites is present in HPV16-immortalized keratinocytes, and this effect seems to be linked to the overexpression of mRNA for this receptor rather than to differences in the surface/internalized ratio of the receptors. ET-1 induces significant increases in [3H]thymidine incorporation and cell proliferation. Furthermore, HPV-transfected keratinocytes can proliferate in the absence of any growth factor added to the growth medium, and the ETA receptor antagonist BQ123 prevents this proliferation. These data suggest a new mechanism in the growth control of HPV-transformed cells mediated by the upregulation of ET-1 autocrine loop.

  18. Dyskeratosis Congenita Dermal Fibroblasts are Defective in Supporting the Clonogenic Growth of Epidermal Keratinocytes

    PubMed Central

    Buckingham, Erin M.; Goldman, Frederick D.; Klingelhutz, Aloysius J.

    2012-01-01

    Telomere shortening is associated with cellular senescence and aging. Dyskeratosis congenita (DC) is a premature aging syndrome caused by mutations in genes for telomerase components or telomere proteins. DC patients have very short telomeres and exhibit aging-associated pathologies including epidermal abnormalities and bone marrow failure. Here, we show that DC skin fibroblasts are defective in their ability to support the clonogenic growth of epidermal keratinocytes. Conditioned media transfer experiments demonstrated that this defect was largely due to lack of a factor or factors secreted from the DC fibroblasts. Compared to early passage normal fibroblasts, DC fibroblasts express significantly lower transcript levels of several genes that code for secreted proteins, including Insulin-like Growth Factor 1 (IGF1) and Hepatocyte Growth Factor (HGF). Aged normal fibroblasts with short telomeres also had reduced levels of IGF1 and HGF, similar to early passage DC fibroblasts. Knockdown of IGF1 or HGF in normal fibroblasts caused a reduction in the capacity of conditioned media from these fibroblasts to support keratinocyte clonogenic growth. Surprisingly, reconstitution of telomerase in DC fibroblasts did not significantly increase transcript levels of IGF1 or HGF or substantially increase the ability of the fibroblasts to support keratinocyte growth, indicating that the gene expression defect is not readily reversible. Our results suggest that telomere shortening in dermal fibroblasts leads to reduction in expression of genes such as IGF1 and HGF and that this may cause a defect in supporting normal epidermal proliferation. PMID:23251848

  19. Platelet-activating factor biosynthesis induced by various stimuli in human HaCaT keratinocytes.

    PubMed

    Travers, J B; Harrison, K A; Johnson, C A; Clay, K L; Morelli, J G

    1996-07-01

    Platelet-activating factor (PAF) is a potent inflammatory mediator that is thought to play a role in cutaneous inflammation. These studies used mass spectrometry to examine the molecular species of PAF precursor glycerophosphocholine lipids (GPC) as well as the biosynthesis of PAF and other sn-2 acetyl-GPC in a human keratinocyte-derived cell line (HaCaT keratinocytes). Approximately 28% of HaCaT keratinocyte GPC consisted of 1-alkyl species, and the relative amounts of the sn-1 alkyl constituents of the PAF precursor 1-alkyl-2-acyl-GPC were as follows: hexadecyl > octadecenyl > octadecyl. Ionophore (A23187)-stimulated HaCaT keratinocytes synthesized both PAF (1-hexadecyl, 1-octadecenyl, and 1-octadecyl species) and less potent 1-acyl analogs (1-palmitoyl, 1-oleoyl, and 1-stearoyl species). PAF production was rapid and maximal by 10 min. The major species of sn-2acetyl-GPC at 2.5 min were 1-hexadecyl-2-acetyl-GPC (2.2 ng/10(6) cells) and 1-palmitoyl-2-acetyl-GPC (2.4 ng/10(6) cells). HaCaT keratinocytes also synthesized PAF and 1-acyl PAF analogs when stimulated with the peptide growth factor endothelin-1 and the nonhydrolyzable PAF receptor agonist carbamyl-PAF. Both 1-hexadecyl-2- acetyl-GPC and 1-palmitoyl-2-acetyl-GPC stimulated intracellular calcium mobilization in HaCaT cells, indicating that these sn-2 acetyl-GPC act in autocrine fashion. These studies revealed that the human keratinocyte-derived cell line HaCaT can synthesize significant amounts of PAF and 1-acyl analogs in vitro from both nonspecific (A23187) and specific (endothelin-1, carbamyl-PAF) stimulation, suggesting a role for this inflammatory lipid mediator in keratinocyte pathophysiology.

  20. Salivary trefoil factor 3 enhances migration of oral keratinocytes.

    PubMed

    Storesund, Trond; Hayashi, Katsuhiko; Kolltveit, Kristin M; Bryne, Magne; Schenck, Karl

    2008-04-01

    Trefoil factor 3 (TFF3) is a member of the mammalian TFF family. Trefoil factors are secreted onto mucosal surfaces of the entire body and exert different effects according to tissue location. Trefoil factors may enhance mucosal healing by modulating motogenic activity, inhibiting apoptosis, and promoting angiogenesis. Trefoil factor 3 is secreted from the submandibular gland and is present in whole saliva. The aim of this study was to assess the migratory and proliferative effects of TFF3 on primary oral human keratinocytes and oral cancer cell lines. The addition of TFF3 increased the migration of both normal oral keratinocytes and the cancer cell line D12, as evaluated by a two-dimensional scratch assay. By contrast, no increase in proliferation or energy metabolism was observed after stimulation with TFF3. Trefoil factor 3-enhanced migration was found to be driven partly by the extracellular signal-related kinase (Erk1/2) pathway, as shown by addition of the mitogen-activated protein kinase (MAPK) inhibitor PD 98059. Previous functional studies on trefoil peptides have all been based on cells from monolayered epithelium like the intestinal mucosa; this is the first report to show that normal and cancerous keratinocytes from stratified epithelium respond to TFF stimuli. Taken together, salivary TFF3 is likely to contribute to oral wound healing. PMID:18353006

  1. Salivary trefoil factor 3 enhances migration of oral keratinocytes.

    PubMed

    Storesund, Trond; Hayashi, Katsuhiko; Kolltveit, Kristin M; Bryne, Magne; Schenck, Karl

    2008-04-01

    Trefoil factor 3 (TFF3) is a member of the mammalian TFF family. Trefoil factors are secreted onto mucosal surfaces of the entire body and exert different effects according to tissue location. Trefoil factors may enhance mucosal healing by modulating motogenic activity, inhibiting apoptosis, and promoting angiogenesis. Trefoil factor 3 is secreted from the submandibular gland and is present in whole saliva. The aim of this study was to assess the migratory and proliferative effects of TFF3 on primary oral human keratinocytes and oral cancer cell lines. The addition of TFF3 increased the migration of both normal oral keratinocytes and the cancer cell line D12, as evaluated by a two-dimensional scratch assay. By contrast, no increase in proliferation or energy metabolism was observed after stimulation with TFF3. Trefoil factor 3-enhanced migration was found to be driven partly by the extracellular signal-related kinase (Erk1/2) pathway, as shown by addition of the mitogen-activated protein kinase (MAPK) inhibitor PD 98059. Previous functional studies on trefoil peptides have all been based on cells from monolayered epithelium like the intestinal mucosa; this is the first report to show that normal and cancerous keratinocytes from stratified epithelium respond to TFF stimuli. Taken together, salivary TFF3 is likely to contribute to oral wound healing.

  2. Role of keratinocyte-derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes.

    PubMed

    Hirobe, Tomohisa

    2005-02-01

    Melanocytes characterized by the activities of tyrosinase, tyrosinase-related protein (TRP)-1 and TRP-2 as well as by melanosomes and dendrites are located mainly in the epidermis, dermis and hair bulb of the mammalian skin. Melanocytes differentiate from melanoblasts, undifferentiated precursors, derived from embryonic neural crest cells. Because hair bulb melanocytes are derived from epidermal melanoblasts and melanocytes, the mechanism of the regulation of the proliferation and differentiation of epidermal melanocytes should be clarified. The regulation by the tissue environment, especially by keratinocytes is indispensable in addition to the regulation by genetic factors in melanocytes. Recent advances in the techniques of tissue culture and biochemistry have enabled us to clarify factors derived from keratinocytes. Alpha-melanocyte-stimulating hormone, adrenocorticotrophic hormone, basic fibroblast growth factor, nerve growth factor, endothelins, granulocyte-macrophage colony-stimulating factor, steel factor, leukemia inhibitory factor and hepatocyte growth factor have been suggested to be the keratinocyte-derived factors and to regulate the proliferation and/or differentiation of mammalian epidermal melanocytes. Numerous factors may be produced in and released from keratinocytes and be involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes through receptor-mediated signaling pathways.

  3. BAG-1 enhances cell-cell adhesion, reduces proliferation and induces chaperone-independent suppression of hepatocyte growth factor-induced epidermal keratinocyte migration

    SciTech Connect

    Hinitt, C.A.M.; Wood, J.; Lee, S.S.; Williams, A.C.; Howarth, J.L.; Glover, C.P.; Uney, J.B.; Hague, A.

    2010-08-01

    Cell motility is important in maintaining tissue homeostasis, facilitating epithelial wound repair and in tumour formation and progression. The aim of this study was to determine whether BAG-1 isoforms regulate epidermal cell migration in in vitro models of wound healing. In the human epidermal cell line HaCaT, endogenous BAG-1 is primarily nuclear and increases with confluence. Both transient and stable p36-Bag-1 overexpression resulted in increased cellular cohesion. Stable transfection of either of the three human BAG-1 isoforms p36-Bag-1 (BAG-1S), p46-Bag-1 (BAG-1M) and p50-Bag-1 (BAG-1L) inhibited growth and wound closure in serum-containing medium. However, in response to hepatocyte growth factor (HGF) in serum-free medium, BAG-1S/M reduced communal motility and colony scattering, but BAG-1L did not. In the presence of HGF, p36-Bag-1 transfectants retained proliferative response to HGF with no change in ERK1/2 activation. However, the cells retained E-cadherin localisation at cell-cell junctions and exhibited pronounced cortical actin. Point mutations in the BAG domain showed that BAG-1 inhibition of motility is independent of its function as a chaperone regulator. These findings are the first to suggest that BAG-1 plays a role in regulating cell-cell adhesion and suggest an important function in epidermal cohesion.

  4. Stimulatory effect of Brazilian propolis on hair growth through proliferation of keratinocytes in mice.

    PubMed

    Miyata, Shota; Oda, Yozo; Matsuo, Chika; Kumura, Haruto; Kobayashi, Ken

    2014-12-10

    Propolis is a natural honeybee hive product with the potential for use in the treatment of dermatological conditions, such as cutaneous abrasions, burns, and acne. In this study, we investigated whether propolis stimulates hair growth in mice. Ethanol-extracted propolis, which contains various physiologically active substances such as caffeic acid and kaempferol, stimulated anagen induction in shaved back skin. Anagen induction occurred without any detectable abnormalities in the shape of the hair follicles (HFs), hair stem cells in the bulge, proliferating hair matrix keratinocytes in the hair bulb, or localization of versican in the dermal papilla. Propolis treatment also stimulated migration of hair matrix keratinocytes into the hair shaft in HFs during late anagen in the depilated back skin. Organotypic culture of skin containing anagen stage HFs revealed significant stimulation of hair matrix keratinocyte proliferation by propolis. Furthermore, propolis facilitated the proliferation of epidermal keratinocytes. These results indicate that propolis stimulates hair growth by inducing hair keratinocyte proliferation.

  5. Stimulatory effect of Brazilian propolis on hair growth through proliferation of keratinocytes in mice.

    PubMed

    Miyata, Shota; Oda, Yozo; Matsuo, Chika; Kumura, Haruto; Kobayashi, Ken

    2014-12-10

    Propolis is a natural honeybee hive product with the potential for use in the treatment of dermatological conditions, such as cutaneous abrasions, burns, and acne. In this study, we investigated whether propolis stimulates hair growth in mice. Ethanol-extracted propolis, which contains various physiologically active substances such as caffeic acid and kaempferol, stimulated anagen induction in shaved back skin. Anagen induction occurred without any detectable abnormalities in the shape of the hair follicles (HFs), hair stem cells in the bulge, proliferating hair matrix keratinocytes in the hair bulb, or localization of versican in the dermal papilla. Propolis treatment also stimulated migration of hair matrix keratinocytes into the hair shaft in HFs during late anagen in the depilated back skin. Organotypic culture of skin containing anagen stage HFs revealed significant stimulation of hair matrix keratinocyte proliferation by propolis. Furthermore, propolis facilitated the proliferation of epidermal keratinocytes. These results indicate that propolis stimulates hair growth by inducing hair keratinocyte proliferation. PMID:25418897

  6. Ratite oils promote keratinocyte cell growth and inhibit leukocyte activation

    PubMed Central

    Bennett, Darin C.; Leung, Gigi; Wang, Eddy; Ma, Sam; Lo, Blanche K. K.; McElwee, Kevin J.; Cheng, Kimberly M.

    2015-01-01

    Traditionally, native Australian aborigines have used emu oil for the treatment of inflammation and to accelerate wound healing. Studies on mice suggest that topically applied emu oil may have anti-inflammatory properties and may promote wound healing. We investigated the effects of ratite oils (6 emu, 3 ostrich, 1 rhea) on immortalized human keratinocytes (HaCaT cells) in vitro by culturing the cells in media with oil concentrations of 0%, 0.5%, and 1.0%. Peking duck, tea tree, and olive oils were used as comparative controls. The same oils at 0.5% concentration were evaluated for their influence on peripheral blood mononuclear cell (PBMC) survival over 48 hr and their ability to inhibit IFNγ production in PBMCs activated by phytohemagglutinin (PHA) in ELISpot assays. Compared to no oil control, significantly shorter population doubling time durations were observed for HaCaT cells cultured in emu oil (1.51 × faster), ostrich oil (1.46 × faster), and rhea oil (1.64 × faster). Tea tree oil demonstrated significant antiproliferative activity and olive oil significantly prolonged (1.35 × slower) cell population doubling time. In contrast, almost all oils, particularly tea tree oil, significantly reduced PBMC viability. Different oils had different levels of inhibitory effect on IFNγ production with individual emu, ostrich, rhea, and duck oil samples conferring full inhibition. This preliminary investigation suggests that emu oil might promote wound healing by accelerating the growth rate of keratinocytes. Combined with anti-inflammatory properties, ratite oil may serve as a useful component in bandages and ointments for the treatment of wounds and inflammatory skin conditions. PMID:26217022

  7. Ratite oils promote keratinocyte cell growth and inhibit leukocyte activation.

    PubMed

    Bennett, Darin C; Leung, Gigi; Wang, Eddy; Ma, Sam; Lo, Blanche K K; McElwee, Kevin J; Cheng, Kimberly M

    2015-09-01

    Traditionally, native Australian aborigines have used emu oil for the treatment of inflammation and to accelerate wound healing. Studies on mice suggest that topically applied emu oil may have anti-inflammatory properties and may promote wound healing. We investigated the effects of ratite oils (6 emu, 3 ostrich, 1 rhea) on immortalized human keratinocytes (HaCaT cells) in vitro by culturing the cells in media with oil concentrations of 0%, 0.5%, and 1.0%. Peking duck, tea tree, and olive oils were used as comparative controls. The same oils at 0.5% concentration were evaluated for their influence on peripheral blood mononuclear cell (PBMC) survival over 48 hr and their ability to inhibit IFNγ production in PBMCs activated by phytohemagglutinin (PHA) in ELISpot assays. Compared to no oil control, significantly shorter population doubling time durations were observed for HaCaT cells cultured in emu oil (1.51×faster), ostrich oil (1.46×faster), and rhea oil (1.64×faster). Tea tree oil demonstrated significant antiproliferative activity and olive oil significantly prolonged (1.35×slower) cell population doubling time. In contrast, almost all oils, particularly tea tree oil, significantly reduced PBMC viability. Different oils had different levels of inhibitory effect on IFNγ production with individual emu, ostrich, rhea, and duck oil samples conferring full inhibition. This preliminary investigation suggests that emu oil might promote wound healing by accelerating the growth rate of keratinocytes. Combined with anti-inflammatory properties, ratite oil may serve as a useful component in bandages and ointments for the treatment of wounds and inflammatory skin conditions. PMID:26217022

  8. Oxidative inhibition of receptor-type protein-tyrosine phosphatase kappa by ultraviolet irradiation activates epidermal growth factor receptor in human keratinocytes.

    PubMed

    Xu, Yiru; Shao, Yuan; Voorhees, John J; Fisher, Gary J

    2006-09-15

    Ultraviolet (UV) irradiation rapidly increases tyrosine phosphorylation (i.e. activates) of epidermal growth factor receptors (EGFR) in human skin. EGFR-dependent signaling pathways drive increased expression of matrix metalloproteinases, whose actions fragment collagen and elastin fibers, the primary structural protein components in skin connective tissue. Connective tissue fragmentation, which results from chronic exposure to solar UV irradiation, is a major determinant of premature skin aging (photoaging). UV irradiation generates reactive oxygen species, which readily react with conserved cysteine residues in the active site of protein-tyrosine phosphatases (PTP). We report here that EGFR activation by UV irradiation results from oxidative inhibition of receptor type PTP-kappa (RPTP-kappa). RPTP-kappa directly counters intrinsic EGFR tyrosine kinase activity, thereby maintaining EGFR in an inactive state. Reversible, oxidative inactivation of RPTP-kappa activity by UV irradiation shifts the kinase-phosphatase balance in favor of EGFR activation. These data delineate a novel mechanism of EGFR regulation and identify RPTP-kappa as a key molecular target for antioxidant protection against skin aging.

  9. Essential role of an activator protein-2 (AP-2)/specificity protein 1 (Sp1) cluster in the UVB-mediated induction of the human vascular endothelial growth factor in HaCaT keratinocytes.

    PubMed Central

    Brenneisen, Peter; Blaudschun, Ralf; Gille, Jens; Schneider, Lars; Hinrichs, Ralf; Wlaschek, Meinhard; Eming, Sabine; Scharffetter-Kochanek, Karin

    2003-01-01

    Chronic sun exposure of the skin has long been postulated to enhance cutaneous angiogenesis, resulting in highly vascularized skin cancers. As the UVB component of sunlight is a major contributor to photocarcinogenesis, we aimed to explore the effects of UVB radiation on vascular endothelial growth factor (VEGF) gene expression, using the immortalized keratinocyte cell line HaCaT as a model for transformed premalignant epithelial cells. In the present paper, we studied the molecular mechanism of UVB-induced VEGF providing a major angiogenic activity in tumour progression and invasion. After 12-24 h of UVB irradiation, a 2.4- to 2.7-fold increase in endogenous VEGF protein level was measured, correlating with an up to 2.5-fold induction of promoter-based reporter gene constructs of VEGF. Furthermore, we identified a GC-rich UVB-responsive region between -87 and -65 bp of the VEGF promoter. In electrophoretic mobility-shift assays, this region binds Sp1-dependent protein complexes constitutively and an additional UVB-inducible protein complex distinct from Sp1 protein. The transcription factor AP-2 (activator protein-2) was detected as a component of the UVB-inducible protein complex. The critical role of the AP-2/Sp1 (specificity protein 1) cluster was supported by demonstration of a significant reduction of UVB-mediated promoter activity upon deletion of this recognition site. The specificity of this region for UVB irradiation was demonstrated using PMA, which increased VEGF activity in HaCaT cells after transient transfection of the deleted promoter construct. In conclusion, our data clarified regulatory mechanisms of UVB-dependent VEGF stimulation which may be critical for angiogenic processes in the skin. PMID:12358602

  10. Expression of paired-like homeodomain transcription factor 2c (PITX2c) in epidermal keratinocytes

    SciTech Connect

    Shi, Ge; Sohn, Kyung-Cheol; Choi, Tae-Young; Choi, Dae-Kyoung; Lee, Sang-Sin; Ou, Bai-sheng; Kim, Sooil; Lee, Young Ho; Yoon, Tae-Jin; Kim, Seong-Jin; Lee, Young; Seo, Young-Joon; Lee, Jeung-Hoon; Kim, Chang Deok

    2010-11-15

    Paired-like homeodomain transcription factor 2 (PITX2) has been implicated as one of the genes responsible for Rieger syndrome. It has been also shown to play a central role during development. In this study, we investigated the functional role of PITX2 in keratinocyte differentiation. RT-PCR analysis showed that PITX2c isoform was predominantly expressed in a differentiation-dependent manner. Consistent with, immunohistochemical staining showed that PITX2 expression was increased in the upper layer of epidermis. When PITX2c was overexpressed in cultured keratinocytes by a recombinant adenovirus, the differentiation markers such as involucrin and loricrin were significantly increased at both mRNA and protein levels. In addition, PITX2c overexpression led to the decrease of cell growth, concomitantly with the upregulation of cell cycle-related genes p21. To investigate the effect of PITX2c in vivo, we microinjected PITX2c expression vector into zebrafish embryo. Interestingly, overexpression of PITX2c in zebrafish embryo led to the formation of horn-like structure and thickening of epidermis, together with the increase of keratin 8 (K8) expression. These results suggest that PITX2c has a role in proliferation and differentiation of epidermal keratinocytes.

  11. Kinetics of growth and differentiation of cultured human epidermal keratinocytes

    SciTech Connect

    Albers, K.M.

    1985-01-01

    A study was made of the interrelationship between replication and differentiation in cultures of human epidermal keratinocytes. Measures of both parameters were made using newly developed methods to quantify the rate at which keratinocytes replicate and the rate at which they withdraw from the cell cycle. Keratinocyte replication was measured by determining the cell doubling time, labeling index, and cell cycle duration. Cell cycle length was measured using a double label assay that determines the length of time between two successive phases of DNA synthesis. The first DNA synthesis phase was marked by labeling keratinocytes with /sup 14/C-thymidine. At the next round of DNA synthesis, cells were labeled with bromodeoxyuridine, a heavy analog of thymidine. The cell cycle length is given by the time required for the /sup 14/C-labeled DNA to become double labeled. To measure keratinocyte differentiation, the rate at which cells withdraw from the cell cycle was determined. To measure withdrawal, the percentage of cells labeled by a pulse of /sup 14/C-thymidine that failed to undergo a second cycle of DNA synthesis, as measured by bromodeoxyuridine incorporation, was determined. Cells which failed to undergo a second cycle of synthesis were considered to have differentiated and withdrawn from the cell cycle.

  12. Growth factors

    SciTech Connect

    Golde, D.W.; Herschman, H.R.; Lusis, A.J.; Groopman, J.E.

    1980-05-01

    Humoral regulation of somatic and hematopoietic cell growth has been intensely investigated during the past decade. Growth hormone is unique because it regulates the size of the person within the constraints of the genetic program. The somatomedins and insulin growth factors are low molecular weight polypeptides believed to mediate some functions of growth hormone. Epithelial growth factor and nerve growth factor are well-characterized polypeptides that influence the growth and differentiation of epithelial and neural tissues and interact with specific cell surface receptors. The hematopoietins are a family of polypeptide hormones that specifically regulate the proliferation and differentiation of stem cells giving rise to erythrocytes, granulocytes, monocytes, megakaryocytes, and B and T lymphocytes. Platelet-derived growth factor modulates the proliferation of fibroblasts in vitro and may have a role in the development of atherosclerosis and myelofibrosis. New knowledge on the biochemistry and physiology of growth factors will probably have a substantial impact on our understanding of human diseases involving abnormal cell growth.

  13. Hair-growth-promoting effect of conditioned medium of high integrin α6 and low CD 71 (α6bri/CD71dim) positive keratinocyte cells.

    PubMed

    Won, Chong Hyun; Jeong, Yun-Mi; Kang, Sangjin; Koo, Tae-Sung; Park, So-Hyun; Park, Ki-Young; Sung, Young-Kwan; Sung, Jong-Hyuk

    2015-01-01

    Keratinocyte stem/progenitor cells (KSCs) reside in the bulge region of the hair follicles and may be involved in hair growth. Hair follicle dermal papilla cells (HFDPCs) and outer root sheath (ORS) cells were treated with conditioned medium (CM) of KSCs. Moreover, the effects of KSC-CM on hair growth were examined ex vivo and in vivo. A human growth factor chip array and RT-PCR were employed to identify enriched proteins in KSC-CM as compared with CM from keratinocytes. KSC-CM significantly increased the proliferation of HFDPCs and ORS cells, and increased the S-phase of the cell cycle in HFDPCs. KSC-CM led to the phosphorylation of ATK and ERK1/2 in both cell types. After subcutaneous injection of KSC-CM in C3H/HeN mice, a significant increase in hair growth and increased proliferation of hair matrix keratinocytes ex vivo was observed. We identified six proteins enriched in KSC-CM (amphiregulin, insulin-like growth factor binding protein-2, insulin-like growth factor binding protein-5, granulocyte macrophage-colony stimulating factor, Platelet-derived growth factor-AA, and vascular endothelial growth factor). A growth-factor cocktail that contains these six recombinant growth factors significantly increased the proliferation of HFDPCs and ORS cells and enhanced the hair growth of mouse models. These results collectively indicate that KSC-CM has the potential to increase hair growth via the proliferative capacity of HFDPCs and ORS cells.

  14. Fish oil constituent docosahexa-enoic acid selectively inhibits growth of human papillomavirus immortalized keratinocytes.

    PubMed

    Chen, D; Auborn, K

    1999-02-01

    The omega-3-fatty acids inhibit proliferation of breast cancer cells whereas omega-6-fatty acids stimulate growth. In this study, we examined effects of these fatty acids on human pre-cancerous cells. Cervical keratinocytes, immortalized with the oncogenic human papillomavirus (HPV) type 16, were treated with linoleic acid, an omega-6-fatty acid, and the omega-3-fatty acids, eicosapentaenoic and docosahexaenoic acids. Using both cell counts and bromodeoxyuridine incorporation, docosahexaenoic acid inhibited growth of these cells to a greater extent than eicosapenta-enoic acid. Linoleic acid had no effect. The effect of docosahexaenoic acid was dose dependent and caused growth arrest. Docosahexaenoic acid inhibited growth of HPV16 immortalized foreskin keratinocytes and laryngeal keratinocytes grown from explants of benign tumors caused by papillomavirus, but had no effect on normal foreskin and laryngeal keratinocytes. Docosahexaenoic acid inhibited growth in the presence of estradiol, a growth stimulator for these cells. Indomethacin, a cyclooxygenase inhibitor like docosahexaenoic acid, had only minimal effect on growth. Alpha-tocopherol, a peroxidation inhibitor, abrogated effects of docosahexaenoic acid implying that inhibitory effects were via lipid peroxidation. PMID:10069461

  15. Snail transcription factors in keratinocytes: Enough to make your skin crawl.

    PubMed

    Sou, Paul W; Delic, Naomi C; Halliday, Gary M; Lyons, J Guy

    2010-12-01

    Keratinocytes are the cells in vertebrates that form the frontline barrier to the environment, and are also the most common origin of human cancer. They normally retain tight cell-cell adhesion and low motility, allowing them to terminally differentiate as they stratify. However, they must be able to respond to tissue damage by migrating into and across wounds. This requires reduced mutual adhesion, suppressed terminal differentiation and increased motility, processes driven by the Snail family of transcriptional repressors. The quantity, location and activity of Snail proteins are regulated by growth factors and cytokines to mediate these responses and invoke an inflammatory response. Subversion of these same pathways can promote carcinoma invasion and metastasis. Signaling network facts: • Snail1 and Snail2 in keratinocytes are important in promoting migration, inflammation and carcinogenesis, and suppressing terminal differentiation. • Extracellular stimuli, including TGFR and EGFR ligands, regulate Snails transcriptionally, via SMAD and MAPK pathways, and post-translationally, by modulating GSK3 and PAK1 activity, which determine Snail stability and intracellular location. • Snails directly repress transcription of genes important for cell-cell adhesion and cornified envelope formation. • Down-regulation of epithelial cadherins by Snails allows LIMDPs to relocate from adherens junctions to the cytoplasm, where they stimulate MAPK pathways, and to the nucleus, where they bind directly to Snails and act as corepressors. • Snail2 is essential for re-epithelialization of healing wounds and can be up-regulated in the keratinocytes at wound margins by p38, ERK1/2 and ERK5 MAPKs, and the arylhydrocarbon receptor. • Further information on signaling related to Snail proteins can be found online at KEGG: http://www.genome.jp/kegg-bin/show pathway?hsa04520 http://www.genome.jp/kegg-bin/show_pathway?hsa04350 http://www.genome.jp/kegg-bin/show pathway?hsa04012

  16. Influence of different buffers (HEPES/MOPS) on keratinocyte cell viability and microbial growth.

    PubMed

    Dias, Kássia de Carvalho; Barbugli, Paula Aboud; Vergani, Carlos Eduardo

    2016-06-01

    This study assessed the effect of the buffers 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 3-(N-morpholino) propanesulfonic acid (MOPS) on keratinocyte cell viability and microbial growth. It was observed that RPMI buffered with HEPES, supplemented with l-glutamine and sodium bicarbonate, can be used as a more suitable medium to promote co-culture. PMID:27060444

  17. Transcription factor 7-like 1 dysregulates keratinocyte differentiation through upregulating lipocalin 2

    PubMed Central

    Xu, M; Zhang, Y; Cheng, H; Liu, Y; Zou, X; Zhan, N; Xiao, S; Xia, Y

    2016-01-01

    Recent studies strongly suggested that transcription factor 7-like 1 (Tcf7l1, also known as Tcf3) is involved in the differentiation of several types of cells, and demonstrated that Tcf7l1 modulates keratinocytes physiologically through regulating lipocalin 2 (LCN2), a key regulator of cell differentiation. To reveal the potential role of Tcf7l1 in the dysregulation of keratinocyte differentiation, both Tcf7l1 and LCN2 were determined in a variety of skin disorders. The in vitro effect of Tcf7l1 on keratinocyte differentiation was studied by culturing SCC-13 cells, and the human foreskin keratinocytes (HFKs) that were transfected with vectors for overexpressing human papillomavirus E6/E7 or Tcf7l1 genes. We found that both Tcf7l1 and LCN2 were highly expressed in those diseases characterized by defective keratinocyte differentiation (especially psoriasis vulgaris, condyloma acuminatum, squamous cell carcinoma, etc). Moreover, compared with control HFKs, SCC-13 cells and E6/E7-harboring HFKs expressed more Tcf7l1 and LCN2. Tcf7l1 siRNA transfection decreased LCN2 but increased involucrin and loricrin in HFKs under calcium stimuli. Conversely, Tcf7l1 overexpression in SCC-13 cells or vector-transfected HFKs induced lower involucrin and loricrin expression and less keratinocyte apoptosis, both of which, however, were partially abrogated by LCN2 siRNA or neutralizing anti-LCN2 antibody. Interestingly, the Tcf7l1 expression in HFKs correlated positively with the MMP-2 level, and the inhibition of MMP-2 decreased the LCN2 level and even attenuated the effect of Tcf7l1 on LCN2 expression. Therefore, Tcf7l1 dysregulates keratinocyte differentiation, possibly through upregulating the LCN2 pathway in an MMP-2 mediated manner. Elucidating the interaction between Tcf7l1 and LCN2 may help understand disordered cell differentiation in some skin diseases. PMID:27551519

  18. Transcription factor 7-like 1 dysregulates keratinocyte differentiation through upregulating lipocalin 2.

    PubMed

    Xu, M; Zhang, Y; Cheng, H; Liu, Y; Zou, X; Zhan, N; Xiao, S; Xia, Y

    2016-01-01

    Recent studies strongly suggested that transcription factor 7-like 1 (Tcf7l1, also known as Tcf3) is involved in the differentiation of several types of cells, and demonstrated that Tcf7l1 modulates keratinocytes physiologically through regulating lipocalin 2 (LCN2), a key regulator of cell differentiation. To reveal the potential role of Tcf7l1 in the dysregulation of keratinocyte differentiation, both Tcf7l1 and LCN2 were determined in a variety of skin disorders. The in vitro effect of Tcf7l1 on keratinocyte differentiation was studied by culturing SCC-13 cells, and the human foreskin keratinocytes (HFKs) that were transfected with vectors for overexpressing human papillomavirus E6/E7 or Tcf7l1 genes. We found that both Tcf7l1 and LCN2 were highly expressed in those diseases characterized by defective keratinocyte differentiation (especially psoriasis vulgaris, condyloma acuminatum, squamous cell carcinoma, etc). Moreover, compared with control HFKs, SCC-13 cells and E6/E7-harboring HFKs expressed more Tcf7l1 and LCN2. Tcf7l1 siRNA transfection decreased LCN2 but increased involucrin and loricrin in HFKs under calcium stimuli. Conversely, Tcf7l1 overexpression in SCC-13 cells or vector-transfected HFKs induced lower involucrin and loricrin expression and less keratinocyte apoptosis, both of which, however, were partially abrogated by LCN2 siRNA or neutralizing anti-LCN2 antibody. Interestingly, the Tcf7l1 expression in HFKs correlated positively with the MMP-2 level, and the inhibition of MMP-2 decreased the LCN2 level and even attenuated the effect of Tcf7l1 on LCN2 expression. Therefore, Tcf7l1 dysregulates keratinocyte differentiation, possibly through upregulating the LCN2 pathway in an MMP-2 mediated manner. Elucidating the interaction between Tcf7l1 and LCN2 may help understand disordered cell differentiation in some skin diseases. PMID:27551519

  19. Hair-Growth-Promoting Effect of Conditioned Medium of High Integrin α6 and Low CD 71 (α6bri/CD71dim) Positive Keratinocyte Cells

    PubMed Central

    Won, Chong Hyun; Jeong, Yun-Mi; Kang, Sangjin; Koo, Tae-Sung; Park, So-Hyun; Park, Ki-Young; Sung, Young-Kwan; Sung, Jong-Hyuk

    2015-01-01

    Keratinocyte stem/progenitor cells (KSCs) reside in the bulge region of the hair follicles and may be involved in hair growth. Hair follicle dermal papilla cells (HFDPCs) and outer root sheath (ORS) cells were treated with conditioned medium (CM) of KSCs. Moreover, the effects of KSC-CM on hair growth were examined ex vivo and in vivo. A human growth factor chip array and RT-PCR were employed to identify enriched proteins in KSC-CM as compared with CM from keratinocytes. KSC-CM significantly increased the proliferation of HFDPCs and ORS cells, and increased the S-phase of the cell cycle in HFDPCs. KSC-CM led to the phosphorylation of ATK and ERK1/2 in both cell types. After subcutaneous injection of KSC-CM in C3H/HeN mice, a significant increase in hair growth and increased proliferation of hair matrix keratinocytes ex vivo was observed. We identified six proteins enriched in KSC-CM (amphiregulin, insulin-like growth factor binding protein-2, insulin-like growth factor binding protein-5, granulocyte macrophage-colony stimulating factor, Platelet-derived growth factor-AA, and vascular endothelial growth factor). A growth-factor cocktail that contains these six recombinant growth factors significantly increased the proliferation of HFDPCs and ORS cells and enhanced the hair growth of mouse models. These results collectively indicate that KSC-CM has the potential to increase hair growth via the proliferative capacity of HFDPCs and ORS cells. PMID:25706512

  20. Cultured human keratinocytes synthesize and secrete endothelin-1.

    PubMed

    Yohn, J J; Morelli, J G; Walchak, S J; Rundell, K B; Norris, D A; Zamora, M R

    1993-01-01

    The human epidermal-melanin unit exists as a complex interplay of cell-cell interactions. Melanocytes synthesize melanin and transfer it to the surrounding keratinocytes, which, in turn, produce factors that affect melanocyte homeostasis, growth, and melanization. Endothelin-1 (ET-1), a vasoconstrictor peptide produced by endothelial cells, has recently been shown to stimulate human melanocyte proliferation and tyrosinase activity. To investigate the possibility that keratinocytes synthesize and secrete ET-1, we grew human keratinocytes in a defined serum-free medium and measured ET-1 levels in the keratinocytes and the keratinocyte-conditioned medium. Northern analysis of keratinocyte total RNA also was performed. We found that human keratinocytes express preproET-1 mRNA and translate the message to ET-1 protein, which is secreted into the keratinocyte medium. Human keratinocytes produced ET-1 in a time-dependent manner with total production of 20.1 +/- 1.1 pg ET-1/10(6) cells at 24 h (n = 7). Although total ET-1 production (secreted plus cell-associated ET-1) was similar, the proportion of secreted versus cell-associated ET-1 varied widely among the different donors. We have found that human keratinocytes synthesize and secrete ET-1 in vitro. From these data we believe that the keratinocyte could be an in vivo epidermal source of this melanocyte growth and pigmentation factor.

  1. Expression of the human papillomavirus type 16 E7 oncoprotein induces an autophagy-related process and sensitizes normal human keratinocytes to cell death in response to growth factor deprivation

    SciTech Connect

    Zhou Xiaobo; Muenger, Karl

    2009-03-01

    Expression of oncogenes, such as the human papillomavirus type 16 (HPV16) E7 oncoprotein, promotes aberrant cell proliferation. In the absence of concurrent mitogenic stimuli, this triggers a cell-intrinsic defense mechanism, the 'trophic sentinel response', which eliminates such aberrant cells. The molecular pathways that elicit this response, however, remain obscure. We set up an experimental system to investigate the trophic sentinel pathway triggered by HPV16 E7 expression in normal human keratinocytes, the natural host cells of HPVs. Keratinocytes expressing HPV16 E7 cultured in E-medium undergo cell death and show increased sub-G1 DNA content when grown to confluence or under conditions of serum deprivation. Moreover, HPV16 E7 expressing human keratinocytes express higher levels of the autophagy marker, LC3-II, which can be abrogated by 3-methyladenine, an autophagy inhibitor. These findings indicate that even under normal culture conditions, HPV16 E7 expression triggers metabolic stress that may result in autophagy, a pathway implicated in carcinogenesis.

  2. Modulation of involucrin and envelope competence in human keratinocytes by hydrocortisone, retinyl acetate, and growth arrest.

    PubMed

    Cline, P R; Rice, R H

    1983-07-01

    Involucrin accumulation and ionophore-assisted envelope formation, markers of keratinocyte differentiation, were found to be highly dependent on culture conditions in the malignant epidermal keratinocyte line, SCC-13, derived from a human squamous cell carcinoma. In confluent cultures, approximately one-half of the cells were competent to form envelopes when grown in medium without hydrocortisone or retinyl acetate supplementation. Addition of hydrocortisone to the medium during growth resulted in up to 90% competence, while addition of retinyl acetate instead resulted in as low as 10% competence. Hydrocortisone partially antagonized the effect of retinyl acetate when both agents were added together. Involucrin levels, measured by radioimmunoassay, were modulated essentially in parallel with envelope competence under the various conditions tested. When the cells were grown in medium supplemented with hydrocortisone, the levels shortly after confluence were over 50-fold higher than in sparse cultures. Regardless of hydrocortisone or retinyl acetate addition, less than 1% of the cells were competent in sparse cultures of growing cells, but up to 90% exhibited this property after growth arrest in serum-free medium containing hydrocortisone. High levels of competence were correlated with cessation of cell division but not with loss of colony-forming efficiency; under optimal conditions, two-thirds of the cells were capable of both envelope formation and colony initiation. Normal human epidermal cells showed a 4- to 5-fold increase in envelope competence from sparse to confluent culture but were insensitive to the suppressive effect of retinyl acetate. The results suggest that some potential differentiated character of malignant keratinocytes may be suppressed in vivo by physiological agents such as vitamin A.

  3. The clinical significance and impact of interleukin 15 on keratinocyte cell growth and migration

    PubMed Central

    Jones, A.M.; Griffiths, J.L.; Sanders, A.J.; Owen, S.; Ruge, F.; Harding, K.G.; Jiang, W.G.

    2016-01-01

    Chronic wounds represent a significant burden to health services and are associated with patient morbidity. Novel methods to diagnose and/or treat problematic wounds are needed. Interleukin (IL)-15 is a cytokine involved in a number of biological processes and disease states such as inflammation, healing and cancer progression. The current study explores the expression profile of IL-15 and IL-15 receptor α (IL-15Rα) in chronic wounds and its impact on keratinocytes. IL-15 and IL-15Rα expression were examined in healing and non-healing chronic wounds using qPCR and immunohistochemical analysis. The impact of recombinant IL-15 (rhIL-15) on human adult low calcium temperature (HaCaT) keratinocyte growth and migratory potential was further examined. IL-15 transcript expression was slightly, though non-significantly elevated in healing chronic wounds compared with non-healing chronic wounds. IL-15 protein staining was minimal in both subtypes of chronic wounds. By contrast, IL-15Rα transcript and protein expression were both observed to be enhanced in non-healing chronic wounds compared with healing chronic wounds. The treatment of HaCaT cells with rhIL-15 generally enhanced cell growth and promoted migration. Analysis with small molecule inhibitors suggested that the pro-migratory effect of rhIL-15 may be associated with ERK, AKT, PLCγ and FAK signalling. IL-15 may promote healing traits in keratinocytes and the differential expression of IL-15Rα is observed in chronic wounds. Together, this may imply a complex role for this interleukin in wound healing. PMID:27460304

  4. Tumor necrosis factor beta and ultraviolet radiation are potent regulators of human keratinocyte ICAM-1 expression

    SciTech Connect

    Krutmann, J.; Koeck, A.S.; Schauer, E.; Parlow, F.; Moeller, A.K.; Kapp, A.; Foerster, E.S.; Schoepf, E.L.; Luger, T.A. )

    1990-08-01

    Intercellular adhesion molecule-1 (ICAM-1) functions as a ligand of leukocyte function-associated antigen-1 (LFA-1), as well as a receptor for human picorna virus, and its regulation thus affects various immunologic and inflammatory reactions. The weak, constitutive ICAM-1 expression on human keratinocytes (KC) can be up-regulated by cytokines such as interferon-gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha). In order to further examine the regulation of KC ICAM-1 expression, normal human KC or epidermoid carcinoma cells (KB) were incubated with different cytokines and/or exposed to ultraviolet (UV) radiation. Subsequently, ICAM-1 expression was monitored cytofluorometrically using a monoclonal anti-ICAM-1 antibody. Stimulation of cells with recombinant human (rh) interleukin (IL) 1 alpha, rhIL-4, rhIL-5, rhIL-6, rh granulocyte/macrophage colony-stimulating factor (GM-CSF), rh interferon alpha (rhIFN alpha), and rh transforming growth factor beta (TGF beta) did not increase ICAM-1 surface expression. In contrast, rhTNF beta significantly up-regulated ICAM-1 expression in a time- and dose-dependent manner. Moreover, the combination of rhTNF beta with rhIFN gamma increased the percentage of ICAM-1-positive KC synergistically. This stimulatory effect of rhTNF beta was further confirmed by the demonstration that rhTNF beta was capable of markedly enhancing ICAM-1 mRNA expression in KC. Finally, exposure of KC in vitro to sublethal doses of UV radiation (0-100 J/m2) prior to cytokine (rhIFN tau, rhTNF alpha, rhTNF beta) stimulation inhibited ICAM-1 up-regulation in a dose-dependent fashion. These studies identify TNF beta and UV light as potent regulators of KC ICAM-1 expression, which may influence both attachment and detachment of leukocytes and possibly viruses to KC.

  5. Lichen metabolites. 2. Antiproliferative and cytotoxic activity of gyrophoric, usnic, and diffractaic acid on human keratinocyte growth.

    PubMed

    Kumar, K C; Müller, K

    1999-06-01

    The sensitivity of the human keratinocyte cell line HaCaT to several lichen metabolites isolated from Parmelia nepalensis and Parmelia tinctorum was evaluated. The tridepside gyrophoric acid (6), the dibenzofuran derivative (+)-usnic acid (1), and the didepside diffractaic acid (5) were potent antiproliferative agents and inhibited cell growth, with IC50 values of 1.7, 2.1, and 2.6 microM, respectively. Methyl beta-orcinolcarboxylate (2), ethyl hematommate (3), the didepside atranorin (4), and (+)-protolichesterinic acid (7) did not influence keratinocyte growth at concentrations of 5 microM. Keratinocytes were further tested for their susceptibility to the action of the potent antiproliferative agents on plasma membrane integrity. The release of lactate dehydrogenase activity into the culture medium was unchanged as compared to controls, documenting that the activity of gyrophoric acid (6), (+)-usnic acid (1), and diffractaic acid (5) was due to cytostatic rather than cytotoxic effects. PMID:10395495

  6. 8-Cl-Adenosine enhances 1,25-dihydroxyvitamin D3-induced growth inhibition without affecting 1,25-dihydroxyvitamin D3-stimulated differentiation of primary mouse epidermal keratinocytes

    PubMed Central

    Bollag, Wendy B; Zhong, Xiaofeng; Josephson, Sarah

    2004-01-01

    Background Epidermal keratinocytes continuously proliferate and differentiate to form the mechanical and water permeability barrier that makes terrestrial life possible. In certain skin diseases, these processes become dysregulated, resulting in abnormal barrier formation. In particular, skin diseases such as psoriasis, actinic keratosis and basal and squamous cell carcinomas are characterized by hyperproliferation and aberrant or absent differentiation of epidermal keratinocytes. We previously demonstrated that 8-Cl-adenosine (8-Cl-Ado) can induce keratinocyte growth arrest without inducing differentiation. Results To determine if this agent might be useful in treating hyperproliferative skin disorders, we investigated whether 8-Cl-Ado could enhance the ability of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], a known keratinocyte differentiating agent and a clinical treatment for psoriasis, to inhibit keratinocyte growth. We found that low concentrations of 8-Cl-Ado and 1,25(OH)2D3 appeared to act additively to reduce proliferation of primary mouse epidermal keratinocytes. However, another agent (transforming growth factor-beta) that triggers growth arrest without inducing differentiation also coincidentally inhibits differentiation elicited by other agents; inhibition of differentiation is suboptimal for treating skin disorders, as differentiation is often already reduced. Thus, we determined whether 8-Cl-Ado also decreased keratinocyte differentiation induced by 1,25(OH)2D3, as measured using the early and late differentiation markers, keratin 1 protein levels and transglutaminase activity, respectively. 8-Cl-Ado did not affect 1,25(OH)2D3-stimulated keratin 1 protein expression or transglutaminase activity. Conclusions Our results suggest that 8-Cl-Ado might be useful in combination with differentiating agents for the treatment of hyperproliferative disorders of the skin. PMID:15279680

  7. Superoxide anions produced by Streptococcus pyogenes group A-stimulated keratinocytes are responsible for cellular necrosis and bacterial growth inhibition.

    PubMed

    Regnier, Elodie; Grange, Philippe A; Ollagnier, Guillaume; Crickx, Etienne; Elie, Laetitia; Chouzenoux, Sandrine; Weill, Bernard; Plainvert, Céline; Poyart, Claire; Batteux, Frédéric; Dupin, Nicolas

    2016-02-01

    Gram-positive Streptococcus pyogenes (group A Streptococcus or GAS) is a major skin pathogen and interacts with keratinocytes in cutaneous tissues. GAS can cause diverse suppurative and inflammatory infections, such as cellulitis, a common acute bacterial dermo-hypodermitis with a high morbidity. Bacterial isolation yields from the lesions are low despite the strong local inflammation observed, raising numerous questions about the pathogenesis of the infection. Using an in vitro model of GAS-infected keratinocytes, we show that the major ROS produced is the superoxide anion ([Formula: see text]), and that its production is time- and dose-dependent. Using specific modulators of ROS production, we show that [Formula: see text] is mainly synthesized by the cytoplasmic NADPH oxidase. Superoxide anion production leads to keratinocyte necrosis but incomplete inhibition of GAS growth, suggesting that GAS may be partially resistant to the oxidative burst. In conclusion, GAS-stimulated keratinocytes are able to develop an innate immune response based on the production of ROS. This local immune response limits GAS development and induces keratinocyte cell death, resulting in the skin lesions observed in patients with cellulitis.

  8. RXRα ablation in epidermal keratinocytes enhances UVR-induced DNA damage, apoptosis, and proliferation of keratinocytes and melanocytes.

    PubMed

    Wang, Zhixing; Coleman, Daniel J; Bajaj, Gaurav; Liang, Xiaobo; Ganguli-Indra, Gitali; Indra, Arup K

    2011-01-01

    We show here that keratinocytic nuclear receptor retinoid X receptor-α (RXRα) regulates mouse keratinocyte and melanocyte homeostasis following acute UVR. Keratinocytic RXRα has a protective role in UVR-induced keratinocyte and melanocyte proliferation/differentiation, oxidative stress-mediated DNA damage, and cellular apoptosis. We discovered that keratinocytic RXRα, in a cell-autonomous manner, regulates mitogenic growth responses in skin epidermis through secretion of heparin-binding EGF-like growth factor, GM-CSF, IL-1α, and cyclooxygenase-2 and activation of mitogen-activated protein kinase pathways. We identified altered expression of several keratinocyte-derived mitogenic paracrine growth factors such as endothelin 1, hepatocyte growth factor, α-melanocyte stimulating hormone, stem cell factor, and fibroblast growth factor-2 in skin of mice lacking RXRα in epidermal keratinocytes (RXRα(ep-/-) mice), which in a non-cell-autonomous manner modulated melanocyte proliferation and activation after UVR. RXRα(ep-/-) mice represent a unique animal model in which UVR induces melanocyte proliferation/activation in both epidermis and dermis. Considered together, the results of our study suggest that RXR antagonists, together with inhibitors of cell proliferation, can be effective in preventing solar UVR-induced photocarcinogenesis.

  9. UV-B radiation induces macrophage migration inhibitory factor-mediated melanogenesis through activation of protease-activated receptor-2 and stem cell factor in keratinocytes.

    PubMed

    Enomoto, Akiko; Yoshihisa, Yoko; Yamakoshi, Takako; Ur Rehman, Mati; Norisugi, Osamu; Hara, Hiroshi; Matsunaga, Kenji; Makino, Teruhiko; Nishihira, Jun; Shimizu, Tadamichi

    2011-02-01

    UV radiation indirectly regulates melanogenesis in melanocytes through a paracrine regulatory mechanism involving keratinocytes. Protease-activated receptor (PAR)-2 activation induces melanosome transfer by increasing phagocytosis of melanosomes by keratinocytes. This study demonstrated that macrophage migration inhibitory factor (MIF) stimulated PAR-2 expression in human keratinocytes. In addition, we showed that MIF stimulated stem cell factor (SCF) release in keratinocytes; however, MIF had no effect on the release of endothelin-1 or prostaglandin E2 in keratinocytes. In addition, MIF had no direct effect on melanin and tyrosinase synthesis in cultured human melanocytes. The effect of MIF on melanogenesis was also examined using a three-dimensional reconstituted human epidermal culture model, which is a novel, commercially available, cultured human epidermis containing functional melanocytes. Migration inhibitory factor induced an increase in melanin content in the epidermis after a 9-day culture period. Moreover, melanin synthesis induced by UV-B stimulation was significantly down-regulated by anti-MIF antibody treatment. An in vivo study showed that the back skin of MIF transgenic mice had a higher melanin content than that of wild-type mice after 12 weeks of UV-B exposure. Therefore, MIF-mediated melanogenesis occurs mainly through the activation of PAR-2 and SCF expression in keratinocytes after exposure to UV-B radiation.

  10. Wounding activates p38 map kinase and activation transcription factor 3 in leading keratinocytes.

    PubMed

    Harper, Erin G; Alvares, Stacy M; Carter, William G

    2005-08-01

    Quiescent epidermis anchors to laminin 5 in the basement membrane via integrin alpha6beta4. Wounding elevates expression of laminin 5, generating leading keratinocytes (LKs) that migrate via beta1 integrins. Laminin 5 was evaluated as a regulator of cell signaling, and mRNA and protein expression in LKs. An in vitro wound model was developed based on suspension and re-adhesion of quiescent human keratinocytes (HKs). DNA microarrays identified multiple mRNAs elevated 1.5 hours after suspension and re-adhesion including activation transcription factor 3 (ATF3). In vitro and in vivo, levels of ATF3 protein elevate in nuclei of LKs, but not in nuclei of the following cells, 2 hours after suspension or wounding but decline by 12-18 hours post injury. Significantly, null defects in laminin 5 or integrin beta4 that inhibit anchorage chronically elevate ATF3 in vivo. This suggests that adhesion to laminin 5, but not other ligands, suppresses activation. On suspension, ATF3 and other transcripts in the microarrays are elevated by phosphorylated p38 mitogen-activated protein kinase (P-p38), a stress kinase that regulates mRNA and cell motility. Inhibition of P-p38 with SB203580 prevents phosphorylation of ATF2, a transcription factor for ATF3 in LKs. Re-adhesion to laminin 5 via alpha6beta4 dephosphorylates P-p38 and suppresses ATF3 protein relative to cells in suspension. Thus, wounding of quiescent HKs disrupts laminin 5 adhesion to activate p38, generating mRNA transcripts that define LKs. Adhesion to deposits of laminin 5 via alpha6beta4 suppresses P-p38 and activation mRNAs including ATF3. Defects in laminin 5 and alpha6beta4 sustain P-p38 with probable pathological effects on transcription and migration.

  11. Opposing Growth Regulatory Roles of Protein Kinase D Isoforms in Human Keratinocytes*

    PubMed Central

    Ryvkin, Vladislav; Rashel, Mohammad; Gaddapara, Trivikram; Ghazizadeh, Soosan

    2015-01-01

    PKD is a family of three serine/threonine kinases (PKD-1, -2, and -3) involved in the regulation of diverse biological processes including proliferation, migration, secretion, and cell survival. We have previously shown that despite expression of all three isoforms in mouse epidermis, PKD1 plays a unique and critical role in wound healing, phorbol ester-induced hyperplasia, and tumor development. In translating our findings to the human, we discovered that PKD1 is not expressed in human keratinocytes (KCs) and there is a divergence in the expression and function of other PKD isoforms. Contrary to mouse KCs, treatment of cultured human KCs with pharmacological inhibitors of PKDs resulted in growth arrest. We found that PKD2 and PKD3 are expressed differentially in proliferating and differentiating human KCs, with the former uniformly present in both compartments whereas the latter is predominantly expressed in the proliferating compartment. Knockdown of individual PKD isoforms in human KCs revealed contrasting growth regulatory roles for PKD2 and PKD3. Loss of PKD2 enhanced KC proliferative potential while loss of PKD3 resulted in a progressive proliferation defect, loss of clonogenicity and diminished tissue regenerative ability. This proliferation defect was correlated with up-regulation of CDK4/6 inhibitor p15INK4B and induction of a p53-independent G1 cell cycle arrest. Simultaneous silencing of PKD isoforms resulted in a more pronounced proliferation defect consistent with a predominant role for PKD3 in proliferating KCs. These data underline the importance and complexity of PKD signaling in human epidermis and suggest a central role for PKD3 signaling in maintaining human epidermal homeostasis. PMID:25802335

  12. Modulation of TGFβ-inducible hypermotility by EGF and other factors in human prostate epithelial cells and keratinocytes

    PubMed Central

    Wei, Wei; Barron, Patricia D.; Rheinwald, James G.

    2013-01-01

    Keratinocytes migrating from a wound edge or initiating malignant invasion greatly increase their expression of the basement membrane protein Laminin-322 (Lam332). In culture, keratinocytes initiate sustained directional hypermotility when plated onto an incompletely processed form of Lam332 (Lam332′) or when treated with TGFβ, an inducer of Lam332 expression. The development and tissue architecture of stratified squamous and prostate epithelia are very different, yet the basal cells of both express p63, α6β4 integrin, and Lam332. Keratinocytes and prostate epithelial cells grow well in nutritionally-optimized culture media with pituitary extract and certain mitogens. We report that prostate epithelial cells display hypermotility responses indistinguishable from those of keratinocytes. Several culture medium variables attenuated TGFβ-induced hypermotility, including Ca++, serum, and some pituitary extract preparations, without impairing growth, TGFβ growth-inhibition, or hypermotility on Lam322′. Distinct from its role as a mitogen, EGF proved to be a required cofactor for TGFβ-induced hypermotility and could not be replaced by HGF or KGF. Prostate epithelial cells have a short replicative lifespan, restricted both by p16INK4A and telomere-related mechanisms. We immortalized the normal prostate epithelial cell line HPrE-1 by transduction to express bmi1 and TERT. Prostate epithelial cells lose expression of p63, β4 integrin, and Lam332 when they transform to invasive carcinoma. In contrast, HPrE-1/bmi1/TERT cells retained expression of these proteins and normal TGFβ signaling and hypermotility for >100 doublings. Thus, keratinocytes and prostate epithelial cells possess common hypermotility and senescence mechanisms and immortalized prostate cell lines can be engineered using defined methods to yield cells retaining normal properties. PMID:21042878

  13. Krüppel-like factor 9 is a circadian transcription factor in human epidermis that controls proliferation of keratinocytes

    PubMed Central

    Spörl, Florian; Korge, Sandra; Jürchott, Karsten; Wunderskirchner, Minetta; Schellenberg, Katja; Heins, Sven; Specht, Aljona; Stoll, Claudia; Klemz, Roman; Maier, Bert; Wenck, Horst; Schrader, Annika; Kunz, Dieter; Blatt, Thomas; Kramer, Achim

    2012-01-01

    Circadian clocks govern a wide range of cellular and physiological functions in various organisms. Recent evidence suggests distinct functions of local clocks in peripheral mammalian tissues such as immune responses and cell cycle control. However, studying circadian action in peripheral tissues has been limited so far to mouse models, leaving the implication for human systems widely elusive. In particular, circadian rhythms in human skin, which is naturally exposed to strong daytime-dependent changes in the environment, have not been investigated to date on a molecular level. Here, we present a comprehensive analysis of circadian gene expression in human epidermis. Whole-genome microarray analysis of suction-blister epidermis obtained throughout the day revealed a functional circadian clock in epidermal keratinocytes with hundreds of transcripts regulated in a daytime-dependent manner. Among those, we identified a circadian transcription factor, Krüppel-like factor 9 (Klf9), that is substantially up-regulated in a cortisol and differentiation-state-dependent manner. Gain- and loss-of-function experiments showed strong antiproliferative effects of Klf9. Putative Klf9 target genes include proliferation/differentiation markers that also show circadian expression in vivo, suggesting that Klf9 affects keratinocyte proliferation/differentiation by controlling the expression of target genes in a daytime-dependent manner. PMID:22711835

  14. Integration of the Transcription Factor-Regulated and Epigenetic Mechanisms in the Control of Keratinocyte Differentiation

    PubMed Central

    Botchkarev, Vladimir A.

    2016-01-01

    The epidermal differentiation program is regulated at several levels including signaling pathways, lineage-specific transcription factors, and epigenetic regulators that establish well-coordinated process of terminal differentiation resulting in formation of the epidermal barrier. The epigenetic regulatory machinery operates at several levels including modulation of covalent DNA/histone modifications, as well as through higher-order chromatin remodeling to establish long-range topological interactions between the genes and their enhancer elements. Epigenetic regulators exhibit both activating and repressive effects on chromatin in keratinocytes (KCs): whereas some of them promote terminal differentiation, the others stimulate proliferation of progenitor cells, as well as inhibit premature activation of terminal differentiation-associated genes. Transcription factor-regulated and epigenetic mechanisms are highly connected, and the p63 transcription factor has an important role in the higher-order chromatin remodeling of the KC-specific gene loci via direct control of the genome organizer Satb1 and ATP-dependent chromatin remodeler Brg1. However, additional efforts are required to fully understand the complexity of interactions between distinct transcription factors and epigenetic regulators in the control of KC differentiation. Further understanding of these interactions and their alterations in different pathological skin conditions will help to progress toward the development of novel approaches for the treatment of skin disorders by targeting epigenetic regulators and modulating chromatin organization in KCs. PMID:26551942

  15. Growth factor signalling.

    PubMed

    de Laat, S W; Boonstra, J; Defize, L H; Kruijer, W; van der Saag, P T; Tertoolen, L G; van Zoelen, E J; den Hertog, J

    1999-01-01

    Signalling between cells in the developing vertebrate embryo is essential for normal embryonic development. In the mid 1970's, signal transduction research started at the Hubrecht Laboratory with special emphasis on analysis of the signalling mechanisms that direct cell proliferation and differentiation. The introduction of in vitro model systems contributed tremendously to the success of the signal transduction research at the Hubrecht Laboratory. Initially neuroblastoma cell lines, and later embryonal carcinoma and embryonal stem cells played an important role in identification of the molecular key players in developmental signalling. For instance, embryonal carcinoma cells were used to identify and characterise polypeptide growth factors. Growth factor signalling research was extended to analysis of growth factor receptor activation. Moreover, the second messenger systems that are linked to growth factor receptors were studied, as well as the nuclear responses to growth factor receptor activation. Finally, the role of growth factor signalling in differentiation was established using embryonal carcinoma cells. Here, we will review work that was characteristic for the growth factor receptor signalling research that was done at the Hubrecht Laboratory between 1980 and the early 1990's.

  16. CORONETTE KERATINOCYTE COLONY FORMATION IS SUPPORTED BY EPIDERMAL-DERMAL CELL INTERACTIONS IN THE BOVINE CLAW

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Delineating factors that orchestrate keratinocyte growth and differentiation in the claw is pivotal to understanding the quality of hoof horn production in health and disease. The specific objectives of this investigation were to establish an in vitro culture system for bovine coronette keratinocyt...

  17. Evidence for multiple bone resorption-stimulating factors produced by normal human keratinocytes in culture.

    PubMed

    Fried, R M; Voelkel, E F; Rice, R H; Levine, L; Tashjian, A H

    1988-06-01

    Conditioned medium from cultured normal human foreskin keratinocytes enhanced the release of calcium from neonatal mouse calvaria in organ culture. Unfractionated keratinocyte-conditioned medium (KCM) stimulated bone resorption in a dose-dependent manner, but it did not increase the concentration of prostaglandin E2 (PGE2) in the bone culture medium until a maximal dose of KCM for resorption was used. Furthermore, inhibitors of PGE2 synthesis, indomethacin, ibuprofen, and piroxicam, did not inhibit KCM-induced calcium release. High concentrations of KCM increased cAMP production by calvaria in the presence of isobutylmethylxanthine, but the increase was small compared with that produced by a dose of bovine PTH that caused a similar level of bone resorption. The bone resorption-stimulating activity of KCM was not lost after incubation at 56 C for 60 min, but it was lost after heating at 100 C for 10 min. Fractionation of KCM by gel filtration chromatography revealed two distinct peaks of bone resorption-stimulating activity. One peak, KCMI, caused a significant increase in bone resorption at 2 micrograms protein/ml. KCMI did not increase medium PGE2, and inhibition of PGE2 synthesis in bone had no effect on KCMI-induced bone resorption. KCMI failed to increase cAMP production by human osteosarcoma SaOS-2 cells. Another peak, KCMII, caused a dose-dependent increase in bone resorption, and a significant increase in medium calcium was noted at a 20-fold lower concentration (0.1 microgram protein/ml) than with KCMI. In contrast to KCMI, the increase in bone resorption stimulated by KCMII was accompanied by a parallel increase in the production of PGE2, and inhibition of PGE2 synthesis completely inhibited the bone resorption-stimulating activity of KCMII. KCMII also caused an increase in cAMP production by SaOS-2 cells. We conclude that KCM contains at least two distinct bone resorption-stimulating factors, one of which acts via a PG-mediated mechanism and the other by

  18. FGF growth factor analogs

    DOEpatents

    Zamora, Paul O.; Pena, Louis A.; Lin, Xinhua; Takahashi, Kazuyuki

    2012-07-24

    The present invention provides a fibroblast growth factor heparin-binding analog of the formula: ##STR00001## where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, X, Y and Z are as defined, pharmaceutical compositions, coating compositions and medical devices including the fibroblast growth factor heparin-binding analog of the foregoing formula, and methods and uses thereof.

  19. Anti-psoriatic drug anthralin activates transcription factor NF-kappa B in murine keratinocytes.

    PubMed

    Schmidt, K N; Podda, M; Packer, L; Baeuerle, P A

    1996-06-01

    Anthralin is one of the most effective and safest therapeutic agents for the treatment of psoriasis, a skin disease characterized by epidermal hyperproliferation and hyperkeratosis. The drug induces and inflammatory response in the skin involving the expression of cytokine and cell adhesion molecule genes that is thought to be essential for its therapeutic efficacy. Reactive oxygen intermediates (ROIs) generated in vivo during the auto-oxidation of anthralin were discussed as mediators of the inflammatory response, but it is not yet understood how this is translated into novel inflammatory gene expression. In this study, we show that at little as 10 microM anthralin can activate a prototypic form of transcription factor NF-(kappa)B, a central transcriptional regulator of inflammatory and immune responses. Two different lines of evidence show that ROIs, in particular H2O2, are second messengers for the anthralin-induced NF-(kappa)B activation. Firstly, the activation could be inhibited by the structurally unrelated antioxidants N-acetyl-L-cysteine and pyrrolidinedithiocarbamate. Secondly, keratinocytes stably overexpressing catalase showed a significant reduction of NF-(kappa)B activation, while stable overexpression of Cu/Zn-superoxide dismutase augmented the anthralin effect. Our data suggest that ROI-induced NF-(kappa)B plays a role in the anti-psoriatic activity of the drug anthralin.

  20. Growth factors for nanobacteria

    NASA Astrophysics Data System (ADS)

    Ciftcioglu, Neva; Kajander, E. Olavi

    1999-12-01

    Nanobacteria are novel microorganisms recently isolated from fetal bovine serum and blood of cows and humans. These coccoid, gram negative bacteria in alpha-2 subgroup of Proteobacteria grow slowly under mammalian cell culture conditions but not in common media for microbes. Now we have found two different kinds of culture supplement preparations that improve their growth and make them culturable in the classical sense. These are supernatant fractions of conditioned media obtained from 1 - 3 months old nanobacteria cultures and from about a 2 weeks old Bacillus species culture. Both improved multiplication and particle yields and the latter increased their resistance to gentamicin. Nanobacteria cultured with any of the methods shared similar immunological property, structure and protein pattern. The growth supporting factors were heat-stabile and nondialyzable, and dialysis improved the growth promoting action. Nanobacteria formed stony colonies in a bacteriological medium supplemented with the growth factors. This is an implication that nanobacterial growth is influenced by pre-existing bacterial flora.

  1. Peptide growth factors, part A

    SciTech Connect

    Barnes, D.; Sirbasku, D.A.

    1987-01-01

    This book contains information on the following topics: Epidermal Growth Factor;Transforming Growth Factors;Bone and Cartilage Growth Factors;Somatomedin/Insulin-Like Growth Factors;Techniques for the Study of Growth Factor Activity;Assays, Phosphorylation, and Surface Membrane Effects.

  2. New microbial growth factor

    NASA Technical Reports Server (NTRS)

    Bok, S. H.; Casida, L. E., Jr.

    1977-01-01

    A screening procedure was used to isolate from soil a Penicillium sp., two bacterial isolates, and a Streptomyces sp. that produced a previously unknown microbial growth factor. This factor was an absolute growth requirement for three soil bacteria. The Penicillium sp. and one of the bacteria requiring the factor, an Arthrobacter sp., were selected for more extensive study concerning the production and characteristics of the growth factor. It did not seem to be related to the siderochromes. It was not present in soil extract, rumen fluid, or any other medium component tested. It appears to be a glycoprotein of high molecular weight and has high specific activity. When added to the diets for a meadow-vole mammalian test system, it caused an increased consumption of diet without a concurrent increase in rate of weight gain.

  3. Enhanced Keratinocyte Proliferation and Migration in Co-culture with Fibroblasts

    PubMed Central

    Wang, Zhenxiang; Wang, Ying; Farhangfar, Farhang; Zimmer, Monica; Zhang, Yongxin

    2012-01-01

    Wound healing is primarily controlled by the proliferation and migration of keratinocytes and fibroblasts as well as the complex interactions between these two cell types. To investigate the interactions between keratinocytes and fibroblasts and the effects of direct cell-to-cell contact on the proliferation and migration of keratinocytes, keratinocytes and fibroblasts were stained with different fluorescence dyes and co-cultured with or without transwells. During the early stage (first 5 days) of the culture, the keratinocytes in contact with fibroblasts proliferated significantly faster than those not in contact with fibroblasts, but in the late stage (11th to 15th day), keratinocyte growth slowed down in all cultures unless EGF was added. In addition, keratinocyte migration was enhanced in co-cultures with fibroblasts in direct contact, but not in the transwells. Furthermore, the effects of the fibroblasts on keratinocyte migration and growth at early culture stage correlated with heparin-binding EGF-like growth factor (HB-EGF), IL-1α and TGF-β1 levels in the cultures where the cells were grown in direct contact. These effects were inhibited by anti-HB-EGF, anti-IL-1α and anti-TGF-β1 antibodies and anti-HB-EGF showed the greatest inhibition. Co-culture of keratinocytes and IL-1α and TGF-β1 siRNA-transfected fibroblasts exhibited a significant reduction in HB-EGF production and keratinocyte proliferation. These results suggest that contact with fibroblasts stimulates the migration and proliferation of keratinocytes during wound healing, and that HB-EGF plays a central role in this process and can be up-regulated by IL-1α and TGF-β1, which also regulate keratinocyte proliferation differently during the early and late stage. PMID:22911722

  4. Keratinocyte dysfunction in vitiligo epidermis: cytokine microenvironment and correlation to keratinocyte apoptosis.

    PubMed

    Moretti, Silvia; Fabbri, Paolo; Baroni, Gianna; Berti, Samantha; Bani, Daniele; Berti, Emilio; Nassini, Romina; Lotti, Torello; Massi, Daniela

    2009-07-01

    Vitiligo is a skin disorder characterized by loss of functional melanocytes. Keratinocytes contribute to melanocyte homeostasis, and keratinocyte alteration may play a role in melanocyte dysfunction in vitiligo. In particular, the release of melanogenic mediators and the level of functioning keratinocytes may affect melanocyte dysfunction in vitiligo epidermis. Keratinocyte-derived mediators involved in pigmentation, analysed by in situ hybridization, and epidermal apoptosis, detected by TUNEL assay and electron microscopy, were evaluated in lesional and perilesional skin biopsies from 15 patients with active vitiligo and in 5 control subjects. Among the melanogenic mediators, stem cell factor (SCF) and endothelin-1 (ET-1) mRNA were significantly reduced in lesional as compared to perilesional epidermis, whereas no difference was observed in mRNA of basic fibroblastic growth factor (bFGF) and granulocyte-monocyte colony stimulating factor (GM-CSF). The expression of mRNA for tumor necrosis factor (TNF)-alpha and interleukin-6 (IL-6), two pro-inflammatory cytokines with an inhibitory effect on pigmentation, was increased in the epidermis from vitiligo biopsies, whereas their expression was practically undetectable in the skin of control subjects. Apoptotic keratinocytes were more abundant in lesional vs. perilesional skin of vitiligo patients and were absent in the epidermis of control subjects. Changes in expression of keratinocyte-derived mediators observed in the present study are consistent with their differential functions in melanocyte regulation. In particular, increased TNF-alpha could contribute to keratinocyte apoptosis, which results in reduced release of melanogenic cytokines and ultimately in melanocyte disappearance.

  5. Peptide growth factors, part B

    SciTech Connect

    Barnes, D.; Sirbasku, D.A.

    1987-01-01

    This book discusses the following topics: Platelet-Derived Growth Factor;Nerve and Glial Growth Factors;PC12 Pheochromocytoma Cells;Techniques for the Study of Growth Factor Activity;Genetic Approaches and Biological Effects.

  6. Epidermal growth factor receptor distribution in burn wounds. Implications for growth factor-mediated repair.

    PubMed Central

    Wenczak, B A; Lynch, J B; Nanney, L B

    1992-01-01

    Epidermal growth factor (EGF) along with several related peptide growth factors has been shown both in vivo and in vitro to accelerate events associated with epidermal wound repair. EGF and transforming growth factor alpha act by binding to a common EGF receptor tyrosine kinase thereby initiating a series of events which ultimately regulate cell proliferation. This study examined the immunohistochemical localization of EGF receptor (EGF-R) in burn wound margins, adjacent proliferating epithelium, and closely associated sweat ducts, sebaceous glands, and hair follicles. Tissue specimens removed during surgical debridement were obtained from full and partial thickness burn wounds in 32 patients with total body surface area burns ranging from 2 to 88%. In the early postburn period (days 2-4), prominent staining for EGF-R was found in undifferentiated, marginal keratinocytes, adjacent proliferating, hypertrophic epithelium, and both marginal and nonmarginal hair follicles, sweat ducts, and sebaceous glands. During the late postburn period (days 5-16), EGF-R was depleted along leading epithelial margins; however, immunoreactive EGF-R remained intensely positive in the hypertrophic epithelium and all skin appendages. Increased detection of immunoreactive EGF-R and the presence of [125I]EGF binding in the hypertrophic epithelium correlated positively with proliferating cell nuclear antigen distributions. Thus, the presence of EGF-R in the appropriate keratinocyte populations suggests a functional role for this receptor during wound repair. Dynamic modulation in EGF receptor distribution during the temporal sequence of repair provides further evidence that an EGF/transforming growth factor alpha/EGF-R-mediated pathway is activated during human wound repair. Images PMID:1361495

  7. Enhanced vascularization of cultured skin substitutes genetically modified to overexpress vascular endothelial growth factor.

    PubMed

    Supp, D M; Supp, A P; Bell, S M; Boyce, S T

    2000-01-01

    Cultured skin substitutes have been used as adjunctive therapies in the treatment of burns and chronic wounds, but they are limited by lack of a vascular plexus. This deficiency leads to greater time for vascularization compared with native skin autografts and contributes to graft failure. Genetic modification of cultured skin substitutes to enhance vascularization could hypothetically lead to improved wound healing. To address this hypothesis, human keratinocytes were genetically modified by transduction with a replication incompetent retrovirus to overexpress vascular endothelial growth factor, a specific and potent mitogen for endothelial cells. Cultured skin substitutes consisting of collagen-glycosaminoglycan substrates inoculated with human fibroblasts and either vascular endothelial growth factor-modified or control keratinocytes were prepared, and were cultured in vitro for 21 d. Northern blot analysis demonstrated enhanced expression of vascular endothelial growth factor mRNA in genetically modified keratinocytes and in cultured skin substitutes prepared with modified cells. Furthermore, the vascular endothelial growth factor-modified cultured skin substitutes secreted greatly elevated levels of vascular endothelial growth factor protein throughout the entire culture period. The bioactivity of vascular endothelial growth factor protein secreted by the genetically modified cultured skin substitutes was demonstrated using a microvascular endothelial cell growth assay. Vascular endothelial growth factor-modified and control cultured skin substitutes were grafted to full-thickness wounds on athymic mice, and elevated vascular endothelial growth factor mRNA expression was detected in the modified grafts for at least 2 wk after surgery. Vascular endothelial growth factor-modified grafts exhibited increased numbers of dermal blood vessels and decreased time to vascularization compared with controls. These results indicate that genetic modification of

  8. NF-κB Protects Human Papillomavirus Type 38 E6/E7-Immortalized Human Keratinocytes against Tumor Necrosis Factor Alpha and UV-Mediated Apoptosis▿

    PubMed Central

    Hussain, Ishraq; Fathallah, Ikbal; Accardi, Rosita; Yue, Jiping; Saidj, Djamel; Shukla, Ruchi; Hasan, Uzma; Gheit, Tarik; Niu, Yamei; Tommasino, Massimo; Sylla, Bakary S.

    2011-01-01

    Constitutive activation of NF-κB signaling is a key event in virus- and non-virus-induced carcinogenesis. We have previously reported that cutaneous human papillomavirus type 38 (HPV38) displays transforming properties in in vitro and in vivo experimental models. However, the involvement of NF-κB signaling in HPV38-induced cell growth transformation remains to be determined. In this study, we showed that HPV38 E6 and E7 activate NF-κB and that inhibition of the pathway with the IκBα superrepressor sensitizes HPV38E6E7-immortalized human keratinocytes to tumor necrosis factor alpha (TNF-α)- and UVB radiation-mediated apoptosis. Accordingly, inhibition of NF-κB signaling resulted in the downregulation of NF-κB-regulated antiapoptotic genes, including cIAP1, cIAP2, and xIAP genes. These findings demonstrate a critical role of NF-κB activity in the survival of HPV38E6E7-immortalized human keratinocytes exposed to cytokine or UV radiation. Our data provide additional evidence for cooperation between beta HPV infection and UV irradiation in skin carcinogenesis. PMID:21715489

  9. Factors affecting ultraviolet-A photon emission from β-irradiated human keratinocyte cells

    NASA Astrophysics Data System (ADS)

    Le, M.; Mothersill, C. E.; Seymour, C. B.; Ahmad, S. B.; Armstrong, A.; Rainbow, A. J.; McNeill, F. E.

    2015-08-01

    The luminescence intensity of 340+/- 5 nm photons emitted from HaCaT (human keratinocyte) cells was investigated using a single-photon-counting system during cellular exposure to 90Y β-particles. Multiple factors were assessed to determine their influence upon the quantity and pattern of photon emission from β-irradiated cells. Exposure of 1× {{10}4} cells/5 mL to 703 μCi resulted in maximum UVA photoemission at 44.8× {{10}3}+/- 2.5× {{10}3} counts per second (cps) from live HaCaT cells (background: 1-5 cps); a 16-fold increase above cell-free controls. Significant biophoton emission was achieved only upon stimulation and was also dependent upon presence of cells. UVA luminescence was measured for 90Y activities 14 to 703 μCi where a positive relationship between photoemission and 90Y activity was observed. Irradiation of live HaCaT cells plated at various densities produced a distinct pattern of emission whereby luminescence increased up to a maximum at 1× {{10}4} cells/5 mL and thereafter decreased. However, this result was not observed in the dead cell population. Both live and dead HaCaT cells were irradiated and were found to demonstrate different rates of photon emission at low β activities (⩽400 μCi). Dead cells exhibited greater photon emission rates than live cells which may be attributable to metabolic processes taking place to modulate the photoemissive effect. The results indicate that photon emission from HaCaT cells is perturbed by external stimulation, is dependent upon the activity of radiation delivered, the density of irradiated cells, and cell viability. It is postulated that biophoton emission may be modulated by a biological or metabolic process.

  10. Factors affecting ultraviolet-A photon emission from β-irradiated human keratinocyte cells.

    PubMed

    Le, M; Mothersill, C E; Seymour, C B; Ahmad, S B; Armstrong, A; Rainbow, A J; McNeill, F E

    2015-08-21

    The luminescence intensity of 340±5 nm photons emitted from HaCaT (human keratinocyte) cells was investigated using a single-photon-counting system during cellular exposure to (90)Y β-particles. Multiple factors were assessed to determine their influence upon the quantity and pattern of photon emission from β-irradiated cells. Exposure of 1 x 10(4) cells/5 mL to 703 μCi resulted in maximum UVA photoemission at 44.8 x 10(3)±2.5 x 10(3) counts per second (cps) from live HaCaT cells (background: 1-5 cps); a 16-fold increase above cell-free controls. Significant biophoton emission was achieved only upon stimulation and was also dependent upon presence of cells. UVA luminescence was measured for (90)Y activities 14 to 703 μCi where a positive relationship between photoemission and (90)Y activity was observed. Irradiation of live HaCaT cells plated at various densities produced a distinct pattern of emission whereby luminescence increased up to a maximum at 1 x 10(4) cells/5 mL and thereafter decreased. However, this result was not observed in the dead cell population. Both live and dead HaCaT cells were irradiated and were found to demonstrate different rates of photon emission at low β activities (⩽400 μCi). Dead cells exhibited greater photon emission rates than live cells which may be attributable to metabolic processes taking place to modulate the photoemissive effect. The results indicate that photon emission from HaCaT cells is perturbed by external stimulation, is dependent upon the activity of radiation delivered, the density of irradiated cells, and cell viability. It is postulated that biophoton emission may be modulated by a biological or metabolic process.

  11. Loss of nuclear receptor RXRα in epidermal keratinocytes promotes the formation of Cdk4-activated invasive melanomas.

    PubMed

    Hyter, Stephen; Bajaj, Gaurav; Liang, Xiaobo; Barbacid, Mariano; Ganguli-Indra, Gitali; Indra, Arup Kumar

    2010-10-01

    Keratinocytes contribute to melanocyte transformation by affecting their microenvironment, in part through the secretion of paracrine factors. Here we report a loss of expression of nuclear receptor RXRα in epidermal keratinocytes during human melanoma progression. In the absence of keratinocytic RXRα, in combination with mutant Cdk4, cutaneous melanoma was generated that metastasized to lymph nodes in a bigenic mouse model. Expression of several keratinocyte-derived mitogenic growth factors (Et-1, Hgf, Scf, α-MSH and Fgf 2 ) was elevated in skin of bigenic mice, whereas Fas, E-cadherin and Pten, implicated in apoptosis, cellular invasion and melanomagenesis, respectively, were downregulated within the microdissected melanocytic tumors. We demonstrated that RXRα is recruited on the proximal promoter of both Et-1 and Hgf, possibly directly regulating their transcription in keratinocytes. These studies demonstrate the contribution of keratinocytic paracrine signaling during the cellular transformation and malignant conversion of melanocytes.

  12. Loss of nuclear receptor RXRα in epidermal keratinocytes promotes the formation of Cdk4-activated invasive melanomas.

    PubMed

    Hyter, Stephen; Bajaj, Gaurav; Liang, Xiaobo; Barbacid, Mariano; Ganguli-Indra, Gitali; Indra, Arup Kumar

    2010-10-01

    Keratinocytes contribute to melanocyte transformation by affecting their microenvironment, in part through the secretion of paracrine factors. Here we report a loss of expression of nuclear receptor RXRα in epidermal keratinocytes during human melanoma progression. In the absence of keratinocytic RXRα, in combination with mutant Cdk4, cutaneous melanoma was generated that metastasized to lymph nodes in a bigenic mouse model. Expression of several keratinocyte-derived mitogenic growth factors (Et-1, Hgf, Scf, α-MSH and Fgf 2 ) was elevated in skin of bigenic mice, whereas Fas, E-cadherin and Pten, implicated in apoptosis, cellular invasion and melanomagenesis, respectively, were downregulated within the microdissected melanocytic tumors. We demonstrated that RXRα is recruited on the proximal promoter of both Et-1 and Hgf, possibly directly regulating their transcription in keratinocytes. These studies demonstrate the contribution of keratinocytic paracrine signaling during the cellular transformation and malignant conversion of melanocytes. PMID:20629968

  13. Mutual induction of growth factor gene expression by epidermal-dermal cell interaction

    PubMed Central

    1993-01-01

    Epithelial-mesenchymal interactions control epidermal growth and differentiation, but little is known about the mechanisms of this interaction. We have examined the effects of human dermal microvascular endothelial cells (DMEC) and fibroblasts on keratinocytes in conventional (feeder layer) and organotypic cocultures (lifted collagen gels) and demonstrated the induction of paracrine growth factor gene expression. Clonal keratinocyte growth was similarly stimulated in cocultures with irradiated DMEC and fibroblasts as feeder cells. This effect is most probably caused by induction of growth factor expression in cocultured dermal cells. Keratinocytes stimulated mRNA levels for KGF and IL-6 in both mesenchymal cell types and GM-CSF in fibroblasts. The feeder effect could not be replaced by conditioned media or addition of isolated growth factors. In organotypic cocultures with keratinocytes growing on collagen gels (repopulated with dermal cells), a virtually normal epidermis was formed within 7 to 10 d. Keratinocyte proliferation was drastically stimulated by dermal cells (histone 3 mRNA expression and BrdU labeling) which continued to proliferate as well in the gel. Expression of all typical differentiation markers was provoked in the reconstituted epithelium, though with different localization as compared to normal epidermis. Keratins K1 and K10 appeared coexpressed but delayed, reflecting conditions in epidermal hyperplasia. Keratin localization and proliferation were normalized under in vivo conditions, i.e., in surface transplants on nude mice. From these data it is concluded that epidermal homeostasis is in part controlled by complex reciprocally induced paracrine acting factors in concert with cell-cell interactions and extracellular matrix influences. PMID:8320264

  14. Characterisation of human fibroblasts as keratinocyte feeder layer using p63 isoforms status.

    PubMed

    Auxenfans, Céline; Thépot, Amélie; Justin, Virginie; Hautefeuille, Agnès; Shahabeddin, Lili; Damour, Odile; Hainaut, Pierre

    2009-01-01

    Large-scale culture of primary keratinocytes allows the production of large epidermal sheet surfaces for the treatment of extensive skin burns. This method is dependent upon the capacity to establish cultures of proliferating keratinocytes in conditions compatible with their clonal expansion while maintaining their capacity to differentiate into the typical squamous pattern of human epidermis. Feeder layers are critical in this process because the fibroblasts that compose this layer serve as a source of adhesion, growth and differentiation factors. In this report, we have characterise the expression patterns of p63 isoforms in primary keratinocytes cultured on two different feeder layer systems, murine 3T3 and human fibroblasts. We show that with the latter, keratinocytes express a higher ratio of Delta N to TAp63 isoform, in relation with higher clonogenic potential. These results indicate that human fibroblasts represent an adequate feeder layer system to support the culture of primary human keratinocytes. PMID:20042803

  15. Ultraviolet radiation-induced apoptosis in keratinocytes: on the role of cytosolic factors.

    PubMed

    Assefa, Zerihun; Van Laethem, An; Garmyn, Marjan; Agostinis, Patrizia

    2005-07-25

    Epidemiological and experimental evidences have established solar ultraviolet (UV) radiation as the leading cause of skin cancers. Specifically, the frequency of non-melanoma skin cancer, one of the malignancies with the most rapidly increasing incidence, is directly related to the total exposure to solar UV light. As part of a general effort to elucidate the components of cellular signal transduction pathways, the mechanisms of cellular responses to UV radiation have received considerable attention over the last few years. These efforts were driven mainly by the conviction that understanding how normal cells respond to extracellular stimuli such as exposure to UV radiation will undoubtedly help in deciphering what goes wrong in a variety of clinical disorders including skin cancers and will assist in the development of novel therapeutic strategies. Studies over the last decade have established that UV radiation induces a bewildering array of signal transduction pathways, some of which could lead to apoptotic cell death. UV-induced cell death by apoptosis is considered to be a natural protective mechanism that removes damaged keratinocytes and circumvents the risk of malignant transformation. In this review, we summarize some of the most important findings regarding the response and role of mitogen-activated protein kinases in UVA and UVB radiation-induced signaling to apoptosis in keratinocytes. We will also briefly discuss what is known about the role of the BCL-2 family of proteins, the emerging role of lysosomal proteases and other important cytosolic signaling proteins in UV-induced apoptosis.

  16. An ideal preparation for dermal regeneration: skin renewal growth factors, the growth factor composites from porcine platelets.

    PubMed

    Wang, Kuo-Hsien; Wu, Yo-Ping Greg; Lo, Wen-Cheng

    2012-12-01

    The use of growth factor composites from platelets has been introduced to many areas of clinical applications and studies. With the richest source of growth factors (GFs), beneficial effects have been shown on tissue regeneration and wound healing. However, animal and clinical studies have revealed inconsistent outcomes with the use of platelet-derived growth factors (PDGFs), which were likely due to variations in the presence and concentrations of GFs between various sources. Autologous PDGFs are considered to be safer, but they are limited by the feasibility of large-scale production to be used extensively in the acute phase, greater surface area, or general cosmetic applications. This study employed a simple process to obtain growth factor composites from activated platelets of porcine origin, namely skin renewal growth factors (SRGF). The functions of SRGF were subsequently evaluated on cultured human fibroblasts, keratinocytes, and melanocytes. Our data revealed that SRGF significantly promoted the proliferation of fibroblasts, accompanied by increased expression of collagens (types I, III, IV, and VIII) and proteoglycans. Diminished proliferation and arrested differentiation of keratinocytes were evidenced by the attenuated expression of laminin V and keratin 10. In addition, SRGF also suppressed the growth of melanocytes and reduced the expression of microphthalmia-associated transcription factor (MITF), tyrosinase, and paired box 3 (PAX3), which mediates melanogensis. Our results suggest that SRGF possesses beneficial properties and is a promising and cost-effective composition for the development of a safe cosmetic agent or topical products for skin regeneration. The development of SRGF may also provide an alternative strategy for tissue engineering.

  17. Plant polyphenols differentially modulate inflammatory responses of human keratinocytes by interfering with activation of transcription factors NF{kappa}B and AhR and EGFR-ERK pathway

    SciTech Connect

    Potapovich, Alla I.; Lulli, Daniela; Fidanza, Paolo; Kostyuk, Vladimir A.; De Luca, Chiara; Pastore, Saveria; Korkina, Liudmila G.

    2011-09-01

    Molecular mechanisms underlying modulation of inflammatory responses in primary human keratinocytes by plant polyphenols (PPs), namely the glycosylated phenylpropanoid verbascoside, the stilbenoid resveratrol and its glycoside polydatin, and the flavonoid quercetin and its glycoside rutin were evaluated. As non-lethal stimuli, the prototypic ligand for epidermal growth factor receptor (EGFR) transforming growth factor alpha (TGFalpha), the combination of tumor necrosis factor (TNFalpha) and interferon (IFNgamma) (T/I), UVA + UVB irradiation, and bacterial lipopolysaccharide (LPS) were used. We demonstrated differential modulation of inflammatory responses in keratinocytes at signal transduction, gene transcription, and protein synthesis levels as a function of PP chemical structure, the pro-inflammatory trigger used, and PP interaction with intracellular detoxifying systems. The PPs remarkably inhibited constitutive, LPS- and T/I-induced but not TGFalpha-induced ERK phosphorylation. They also suppressed NFkappaB activation by LPS and T/I. Verbascoside and quercetin invariably impaired EGFR phosphorylation and UV-associated aryl hydrocarbon receptor (AhR)-mediated signaling, while rutin, polydatin and resveratrol did not affect EGFR phosphorylation and further activated AhR machinery in UV-exposed keratinocytes. In general, PPs down-regulated gene expression of pro-inflammatory cytokines/enzymes, except significant up-regulation of IL-8 observed under stimulation with TGFalpha. Both spontaneous and T/I-induced release of IL-8 and IP-10 was suppressed, although 50 {mu}M resveratrol and polydatin up-regulated IL-8. At this concentration, resveratrol activated both gene expression and de novo synthesis of IL-8 and AhR-mediated mechanisms were involved. We conclude that PPs differentially modulate the inflammatory response of human keratinocytes through distinct signal transduction pathways, including AhR and EGFR. - Graphical abstract: Display Omitted Highlights

  18. Primary structure of keratinocyte transglutaminase

    SciTech Connect

    Phillips, M.A.; Stewart, B.E.; Qin, Q.; Rice, R.H. ); Chakravarty, R. ); Floyd, E.E.; Jetten, A.M. )

    1990-12-01

    The nucleotide and deduced amino acid sequences of the coding regions of human and rat keratinocyte transglutaminases (protein-glutamine: amine {gamma}-glutamyltransferase; EC 2.3.2.13) have been determined. These yield proteins of {approximately}90 kDa that are 92% identical, indicative of the conservation of important structural features. Alignments of amino acid sequences show substantial similarity among the keratinocyte transglutaminase, human clotting factor XIII catalytic subunit, guinea pig liver tissue transglutaminase, and the human erythrocyte band-4.2 protein. The keratinocyte enzyme is most similar to factor XIII, whereas the band-4.2 protein is most similar to the tissue transglutaminase. A salient feature of the keratinocyte transglutaminase is its 105-residue extension beyond the N terminus of the tissue transglutaminase. This extension and the unreltaed activation peptide of factor XIII (a 37-residue extension) appear to be added for specialized functions after divergence of the tissue transglutaminase from their common lineage.

  19. Transforming Growth Factor Beta 3 Is Required for Excisional Wound Repair In Vivo

    PubMed Central

    Le, Mark; Naridze, Rachelle; Morrison, Jasmine; Biggs, Leah C.; Rhea, Lindsey; Schutte, Brian C.; Kaartinen, Vesa; Dunnwald, Martine

    2012-01-01

    Wound healing is a complex process that relies on proper levels of cytokines and growth factors to successfully repair the tissue. Of particular interest are the members of the transforming growth factor family. There are three TGF-ß isoforms–TGF- ß 1, 2, and 3, each isoform showing a unique expression pattern, suggesting that they each play a distinct function during development and repair. Previous studies reported an exclusive role for TGF-ß 3 in orofacial development and a potent anti-scarring effect. However, the role of TGF- ß 3 in excisional wound healing and keratinocyte migration remains poorly understood. We tested the effect of TGF-ß 3 levels on excisional cutaneous wounds in the adult mouse by directly injecting recombinant TGF-ß 3 or neutralizing antibody against TGF-ß 3 (NAB) in the wounds. Our results demonstrate that TGF-ß 3 does not promote epithelialization. However, TGF-ß 3 is necessary for wound closure as wounds injected with neutralizing antibody against TGF-ß 3 showed increased epidermal volume and proliferation in conjunction with a delay in keratinocyte migration. Wild type keratinocytes treated with NAB and Tgfb3-deficient keratinocytes closed an in vitro scratch wound with no delay, suggesting that our in vivo observations likely result from a paracrine effect. PMID:23110169

  20. Upregulation of epidermal growth factor receptor 4 in oral leukoplakia

    PubMed Central

    Kobayashi, Hiroshi; Kumagai, Kenichi; Gotoh, Akito; Eguchi, Takanori; Yamada, Hiroyuki; Hamada, Yoshiki; Suzuki, Satsuki; Suzuki, Ryuji

    2013-01-01

    In the present study, we investigate the expression profile of the epidermal growth factor receptor family, which comprises EGFR/ErbB1, HER2/ErbB2, HER3/ErbB3 and HER4/ErbB4 in oral leukoplakia (LP). The expression of four epidermal growth factor receptor (EGFR) family genes and their ligands were measured in LP tissues from 14 patients and compared with levels in 10 patients with oral lichen planus (OLP) and normal oral mucosa (NOM) from 14 healthy donors by real-time polymerase chain reaction (PCR) and immunohistochemistry. Synchronous mRNA coexpression of ErbB1, ErbB2, ErbB3 and ErbB4 was detected in LP lesions. Out of the receptors, only ErbB4 mRNA and protein was more highly expressed in LP compared with NOM tissues. These were strongly expressed by epithelial keratinocytes in LP lesions, as shown by immunohistochemistry. Regarding the ligands, the mRNA of Neuregulin2 and 4 were more highly expressed in OLP compared with NOM tissues. Therefore, enhanced ErbB4 on the keratinocytes and synchronous modulation of EGFR family genes may contribute to the pathogenesis and carcinogenesis of LP. PMID:23492901

  1. Keratinocyte-releasable factors increased the expression of MMP1 and MMP3 in co-cultured fibroblasts under both 2D and 3D culture conditions.

    PubMed

    Li, Min; Moeen Rezakhanlou, Alireza; Chavez-Munoz, Claudia; Lai, Amy; Ghahary, Aziz

    2009-12-01

    Matrix metalloproteinases (MMPs) are key elements in extracellular matrix (ECM) degradation and scar remodeling during the wound-healing process. Our previous data revealed that keratinocyte-releasable factors significantly increased the expression of fibroblast MMPs in monolayer-cultured fibroblasts. In this study, we analyzed the differences in the MMP expressions of fibroblasts in a three-dimensional fibroblast-populated collagen gel (3D FPCG) from that in a two-dimensional monolayer-cultured fibroblasts when both co-cultured with keratinocytes. Differential mRNA and protein expression of fibroblasts were examined by microarray, RT-PCR, and western blot. Our results showed that fibroblasts co-cultured with keratinocytes in a 3D FPCG expressed significantly higher MMP1 and MMP3 at the gene and protein levels. Due to the physiological advantages of a 3D FPCG model to a 2D system, we concluded that the 3D FPCG model may provide a better means of understanding the fibroblast-keratinocyte cross-talk during the wound-healing process. PMID:19521668

  2. Oncogenes, genes, and growth factors

    SciTech Connect

    Guroff, G.

    1989-01-01

    This book contains 12 chapters. Some of the chapter titles are: The Epidermal Growth Factor Receptor Gene; Structure and Expression of the Nerve Growth Factor Gene; The Erythropoietin Gene; The Interleukin-2 Gene; The Transferrin Gene; and The Transferrin Receptor Gene.

  3. Regulation of adrenomedullin secretion in cultured human skin and oral keratinocytes.

    PubMed

    Kapas, S; Tenchini, M L; Farthing, P M

    2001-08-01

    Adrenomedullin, a potent vasoactive peptide, is actively secreted from primary cultures of human oral and skin keratinocytes, but nothing is known of the regulation of its release. This study describes the effects of a range of substances on adrenomedullin production from cultures of oral and skin keratinocytes. We have established that keratinocytes do not store adrenomedullin but secrete it constitutively. Cytokines interleukin-1alpha and -1beta, tumor necrosis factor-alpha and -beta, and the bacterial product, lipopolysaccharide, significantly stimulate adrenomedullin secretion from oral but not skin keratinocytes. Both transforming growth factor-beta1 and interferon-gamma are potent suppressors of adrenomedullin secretion from both cell types, as are forskolin, di-butyryl cyclic adenosine monophosphate, and adrenocorticotropin. The peptides thrombin and endothelin-1 increase adrenomedullin production, particularly from skin keratinocytes. These findings indicate that there are differences in the regulation of adrenomedullin production between oral and skin keratinocytes and that oral keratinocytes are particularly responsive to the action of inflammatory cytokines. This raises the possibility that adrenomedullin may serve a different functions in oral mucosa and skin.

  4. Cyclic stretch induces upregulation of endothelin-1 with keratinocytes in vitro: possible role in mechanical stress-induced hyperpigmentation.

    PubMed

    Kurita, Masakazu; Okazaki, Mutsumi; Fujino, Takashi; Takushima, Akihiko; Harii, Kiyonori

    2011-05-27

    The aim of this study was to investigate the possible pathological relation between mechanical stress and hyperpigmentation. We did this by investigating the influence of cyclic stretch on the expression of keratinocyte- and fibroblast-derived melanogenetic paracrine cytokines in vitro. Using primary human keratinocytes and fibroblasts, alterations of mRNA expression of melanogenetic paracrine cytokines due to cyclic stretch were investigated using a real-time polymerase chain reaction (PCR). The cytokines included basic fibroblast growth factor (bFGF), stem cell factor (SCF), granulocyte/macrophage colony-stimulating factor, interleukin-1α, and endothelin-1 (ET-1) for keratinocytes and bFGF, SCF, and hepatocyte growth factor for fibroblasts. The dose dependence of keratinocyte-derived ET-1 upregulation was further investigated using real-time PCR and an enzyme-linked immunosorbent assay. We also investigated the effects of cyclic stretch on the proliferation and differentiation of keratinocytes. Among the melanogenetic paracrine cytokines investigated, keratinocyte-derived ET-1 was consistently upregulated in all four cell lines. The degree of upregulation increased with the degree of the length and frequency of the stretch; in contrast, cell number and differentiation markers showed no obvious alterations with cyclic stretch. Keratinocyte-derived ET-1 upregulation possibly plays a significant role in the pathogenesis of pigmented disorders, such as friction melanosis, caused by mechanical stress.

  5. Effects of cerium oxide nanoparticles on the growth of keratinocytes, fibroblasts and vascular endothelial cells in cutaneous wound healing.

    PubMed

    Chigurupati, Srinivasulu; Mughal, Mohamed R; Okun, Eitan; Das, Soumen; Kumar, Amit; McCaffery, Michael; Seal, Sudipta; Mattson, Mark P

    2013-03-01

    Rapid and effective wound healing requires a coordinated cellular response involving fibroblasts, keratinocytes and vascular endothelial cells (VECs). Impaired wound healing can result in multiple adverse health outcomes and, although antibiotics can forestall infection, treatments that accelerate wound healing are lacking. We now report that topical application of water soluble cerium oxide nanoparticles (Nanoceria) accelerates the healing of full-thickness dermal wounds in mice by a mechanism that involves enhancement of the proliferation and migration of fibroblasts, keratinocytes and VECs. The Nanoceria penetrated into the wound tissue and reduced oxidative damage to cellular membranes and proteins, suggesting a therapeutic potential for topical treatment of wounds with antioxidant nanoparticles.

  6. Interleukin-1 of cholesteatomatous keratinocytes.

    PubMed

    Kakiuchi, H; Kinoshita, K; Katoh, Y; Tabata, T

    1992-10-01

    Interleukin-1 (IL-1) has been thought to be one of the essential cytokines mainly produced by macrophages. It has recently been reported that epidermal keratinocytes produce IL-1, and attention is being paid to local immune reactions mediated with this cytokine. Interleukin-1 not only activates lymphocytes, but also acts as an osteoclast-activating factor. In this study, we used immunohistochemistry and immunoblotting on cholesteatomatous epithelium with anti-IL-1 alpha antibody and anti-IL-1 beta antibody. Next, the relationship of cholesteatomatous debris to the production of IL-1 by keratinocytes was evaluated. Highly concentrated IL-1 alpha was found in the cholesteatomatous epithelium, especially in the basal cell layer. The intensity of IL-1 beta staining was weaker than that of IL-1 alpha staining. In the immunoblotting study, the 31 kd band, an intracellular immature precursor molecule, was identified. The production of IL-1 alpha from keratinocytes was augmented to a greater degree by cholesteatomatous debris than by lipopolysaccharide or keratin. The keratinocytes did not produce IL-1 beta. These findings suggest that IL-1 alpha is derived from cholesteatomatous keratinocytes. Interleukin-1, mainly IL-1 alpha, from the stimulated cholesteatomatous keratinocytes may be an important factor in the markedly increased bone resorption observed in cholesteatoma.

  7. Galectin-7 regulates keratinocyte proliferation and differentiation through JNK-miR-203-p63 signaling

    PubMed Central

    Chen, Hung-Lin; Chiang, Po-Cheng; Lo, Chia-Hui; Lo, Yuan-Hsin; Hsu, Daniel K.; Chen, Huan-Yuan; Liu, Fu-Tong

    2015-01-01

    Galectin-7, a member of the β-galactoside-binding protein family, is primarily expressed in stratified epithelial cells, including keratinocytes. There is information in the literature suggesting a role for this protein in regulation of keratinocyte survival and growth, but the underlying mechanism remains relatively unknown. Moreover, its expression pattern in the epidermis suggests that it is also involved in the regulation of keratinocyte differentiation. Here, we demonstrate that galectin-7 knockdown results in reduced differentiation and increased proliferation of keratinocytes. Using microarray and deep-sequencing analyses, we found that galectin-7 positively and negatively regulates microRNA (miR)-203 and miR-146a expression, respectively. We show that galectin-7 regulates keratinocyte differentiation and proliferation through miR-203 but not miR-146a. A knockdown of either galectin-7 or miR-203 in keratinocytes increases expression of p63, an essential transcription factor involved in skin development. Rescue of miR-203 expression in a galectin-7 knockdown model reduces p63 expression to baseline. Increased galectin-7 expression up-regulates c-Jun N-terminal kinase (JNK) protein levels, which is required for miR-203 expression. Finally, we establish that galectin-7 can be associated with JNK1 and protect it from ubiquitination and degradation. Thus, our data suggest an intracellular function of galectin-7: regulation of keratinocyte proliferation and differentiation through the JNK1-miR-203-p63 pathway. PMID:26763438

  8. Galectin-7 Regulates Keratinocyte Proliferation and Differentiation through JNK-miR-203-p63 Signaling.

    PubMed

    Chen, Hung-Lin; Chiang, Po-Cheng; Lo, Chia-Hui; Lo, Yuan-Hsin; Hsu, Daniel K; Chen, Huan-Yuan; Liu, Fu-Tong

    2016-01-01

    Galectin-7, a member of the β-galactoside-binding protein family, is primarily expressed in stratified epithelial cells, including keratinocytes. There is information in the literature suggesting a role for this protein in regulation of keratinocyte survival and growth, but the underlying mechanism remains relatively unknown. Moreover, its expression pattern in the epidermis suggests that it is also involved in the regulation of keratinocyte differentiation. Here, we demonstrate that galectin-7 knockdown results in reduced differentiation and increased proliferation of keratinocytes. Using microarray and deep-sequencing analyses, we found that galectin-7 positively and negatively regulates microRNA (miR)-203 and miR-146a expression, respectively. We show that galectin-7 regulates keratinocyte differentiation and proliferation through miR-203 but not miR-146a. A knockdown of either galectin-7 or miR-203 in keratinocytes increases expression of p63, an essential transcription factor involved in skin development. Rescue of miR-203 expression in a galectin-7 knockdown model reduces p63 expression to baseline. Increased galectin-7 expression upregulates c-Jun N-terminal kinase (JNK) protein levels, which is required for miR-203 expression. Finally, we establish that galectin-7 can be associated with JNK1 and protect it from ubiquitination and degradation. Thus, our data suggest an intracellular function of galectin-7: regulation of keratinocyte proliferation and differentiation through the JNK1-miR-203-p63 pathway.

  9. Interstitial fibrosis and growth factors.

    PubMed Central

    Lasky, J A; Brody, A R

    2000-01-01

    Interstitial pulmonary fibrosis (IPF) is scarring of the lung caused by a variety of inhaled agents including mineral particles, organic dusts, and oxidant gases. The disease afflicts millions of individuals worldwide, and there are no effective therapeutic approaches. A major reason for this lack of useful treatments is that few of the molecular mechanisms of disease have been defined sufficiently to design appropriate targets for therapy. Our laboratory has focused on the molecular mechanisms through which three selected peptide growth factors could play a role in the development of IPF. Hundreds of growth factors and cytokines could be involved in the complex disease process. We are studying platelet-derived growth factor because it is the most potent mesenchymal cell mitogen yet described, transforming growth factor beta because it is a powerful inducer of extracellular matrix (scar tissue) components by mesenchymal cells, and tumor necrosis factor alpha because it is a pleiotropic cytokine that we and others have shown is essential for the development of IPF in animal models. This review describes some of the evidence from studies in humans, in animal models, and in vitro, that supports the growth factor hypothesis. The use of modern molecular and transgenic technologies could elucidate those targets that will allow effective therapeutic approaches. Images Figure 1 Figure 2 PMID:10931794

  10. Overexpression of CRABPI in suprabasal keratinocytes enhances the proliferation of epidermal basal keratinocytes in mouse skin topically treated with all-trans retinoic acid

    SciTech Connect

    Tang, X.-H.; Vivero, Marina; Gudas, Lorraine J.

    2008-01-01

    We investigated whether ectopic expression of CRABPI, a cellular retinoic acid binding protein, influenced the actions of all-trans retinoic acid (ATRA) in transgenic (TG) mice. We targeted CRABPI to the basal vs. suprabasal layers of mouse epidermis by using the keratin 14 (K14) and keratin 10 (K10) promoters, respectively. Greater CRABPI protein levels were detected in the epidermis of adult transgenic(+) mice than in transgenic(-) mice for both transgenes. In adult mouse skin CRABPI overexpression in the basal or suprabasal keratinocytes did not cause morphological abnormalities, but did result in decreased CRABPII mRNA levels. Ectopically overexpressed CRABPI in suprabasal keratinocytes, but not in basal keratinocytes, enhanced the thickening of the epidermis induced by topical ATRA treatments (10 {mu}M, 400 {mu}l for 4 days) by 1.59 {+-} 0.2-fold (p < 0.05). ATRA treatment (10 {mu}M) resulted in a 59.9 {+-} 9.8% increase (p < 0.05) in the BrdU labeling index in K10/FLAG-CRABPI TG(+) mice vs. TG(-) mice. Retinoid topical treatments reduced p27 and CYP26A1 mRNA levels in TG(+) and TG(-) mouse skin in K14 and K10/FLAG-CRABPI transgenic mice. As epidermal basal keratinocyte proliferation is stimulated by paracrine growth factors secreted by ATRA activated suprabasal keratinocytes, our results indicate that CRABPI overexpression in suprabasal keratinocytes enhances the physiological functions of ATRA.

  11. Human keratinocytes are a source for tumor necrosis factor alpha: Evidence for synthesis and release upon stimulation with endotoxin or ultraviolet light

    SciTech Connect

    Koeck, A.S.; Schwarz, T.; Kirnbauer, R.; Urbanski, A.; Perry, P.; Ansel, J.C.; Luger, T.A. )

    1990-12-01

    Tumor necrosis factor alpha (TNF-alpha), in addition to being cytotoxic for certain tumor cells, has turned out as a multifunctional cytokine that is involved in the regulation of immunity and inflammation. Since human keratinocytes have been demonstrated to be a potent source of various cytokines, it was investigated whether epidermal cells synthesize and release TNF-alpha. Supernatants derived from normal human keratinocytes (HNK) and human epidermoid carcinoma cell lines (KB, A431) were tested both in a TNF-alpha-specific ELISA and a bioassay. In supernatants of untreated epidermal cells, no or minimal TNF-alpha activity was found, while after stimulation with lipopolysaccharide (LPS) or ultraviolet (UV) light, significant amounts were detected. Western blot analysis using an antibody directed against human TNF-alpha revealed a molecular mass of 17 kD for keratinocyte-derived TNF-alpha. These biological and biochemical data were also confirmed by Northern blot analysis revealing mRNA specific for TNF-alpha in LPS- or ultraviolet B (UVB)-treated HNK and KB cells. In addition, increased TNF-alpha levels were detected in the serum obtained from human volunteers 12 and 24 h after a single total body UVB exposure, which caused a severe sunburn reaction. These findings indicate that keratinocytes upon stimulation are able to synthesize and release TNF-alpha, which may gain access to the circulation. Thus, TNF-alpha in concert with other epidermal cell-derived cytokines may mediate local and systemic inflammatory reactions during host defense against injurious events caused by microbial agents or UV irradiation.

  12. Contribution of Sp1 to Telomerase Expression and Activity in Skin Keratinocytes Cultured With a Feeder Layer.

    PubMed

    Bisson, Francis; Paquet, Claudie; Bourget, Jean-Michel; Zaniolo, Karine; Rochette, Patrick J; Landreville, Solange; Damour, Odile; Boudreau, François; Auger, François A; Guérin, Sylvain L; Germain, Lucie

    2015-02-01

    The growth of primary keratinocytes is improved by culturing them with a feeder layer. The aim of this study was to assess whether the feeder layer increases the lifespan of cultured epithelial cells by maintaining or improving telomerase activity and expression. The addition of an irradiated fibroblast feeder layer of either human or mouse origin (i3T3) helped maintain telomerase activity as well as expression of the transcription factor Sp1 in cultured keratinocytes. In contrast, senescence occurred earlier, together with a reduction of Sp1 expression and telomerase activity, in keratinocytes cultured without a feeder layer. Telomerase activity was consistently higher in keratinocytes grown on the three different feeder layers tested relative to cells grown without them. Suppression of Sp1 expression by RNA inhibition (RNAi) reduced both telomerase expression and activity in keratinocytes and also abolished their long-term growth capacity suggesting that Sp1 is a key regulator of both telomerase gene expression and cell cycle progression of primary cultured human skin keratinocytes. The results of the present study therefore suggest that the beneficial influence of the feeder layer relies on its ability to preserve telomerase activity in cultured human keratinocytes through the maintenance of stable levels of Sp1 expression.

  13. Contribution of Sp1 to Telomerase Expression and Activity in Skin Keratinocytes Cultured With a Feeder Layer.

    PubMed

    Bisson, Francis; Paquet, Claudie; Bourget, Jean-Michel; Zaniolo, Karine; Rochette, Patrick J; Landreville, Solange; Damour, Odile; Boudreau, François; Auger, François A; Guérin, Sylvain L; Germain, Lucie

    2015-02-01

    The growth of primary keratinocytes is improved by culturing them with a feeder layer. The aim of this study was to assess whether the feeder layer increases the lifespan of cultured epithelial cells by maintaining or improving telomerase activity and expression. The addition of an irradiated fibroblast feeder layer of either human or mouse origin (i3T3) helped maintain telomerase activity as well as expression of the transcription factor Sp1 in cultured keratinocytes. In contrast, senescence occurred earlier, together with a reduction of Sp1 expression and telomerase activity, in keratinocytes cultured without a feeder layer. Telomerase activity was consistently higher in keratinocytes grown on the three different feeder layers tested relative to cells grown without them. Suppression of Sp1 expression by RNA inhibition (RNAi) reduced both telomerase expression and activity in keratinocytes and also abolished their long-term growth capacity suggesting that Sp1 is a key regulator of both telomerase gene expression and cell cycle progression of primary cultured human skin keratinocytes. The results of the present study therefore suggest that the beneficial influence of the feeder layer relies on its ability to preserve telomerase activity in cultured human keratinocytes through the maintenance of stable levels of Sp1 expression. PMID:24962522

  14. Ethanol Extract of Cirsium japonicum var. ussuriense Kitamura Exhibits the Activation of Nuclear Factor Erythroid 2-Related Factor 2-dependent Antioxidant Response Element and Protects Human Keratinocyte HaCaT Cells Against Oxidative DNA Damage

    PubMed Central

    Yoo, Ok-Kyung; Choi, Bu Young; Park, Jin-Oh; Lee, Ji-Won; Park, Byoung-Kwon; Joo, Chul Gue; Heo, Hyo-Jung; Keum, Young-Sam

    2016-01-01

    Keratinocytes are constantly exposed to extracellular insults, such as ultraviolet B, toxic chemicals and mechanical stress, all of which can facilitate the aging of keratinocytes via the generation of intracellular reactive oxygen species (ROS). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that plays a critical role in protecting keratinocytes against oxidants and xenobiotics by binding to the antioxidant response element (ARE), a cis-acting element existing in the promoter of most phase II cytoprotective genes. In the present study, we have attempted to find novel ethanol extract(s) of indigenous plants of Jeju island, Korea that can activate the Nrf2/ARE-dependent gene expression in human keratinocyte HaCaT cells. As a result, we identified that ethanol extract of Cirsium japonicum var. ussuriense Kitamura (ECJUK) elicited strong stimulatory effect on the ARE-dependent gene expression. Supporting this observation, we found that ECJUK induced the expression of Nrf2, hemoxygenase-1, and NAD(P)H:quinone oxidoreductase-1 and this event was correlated with Akt1 phosphorylation. We also found that ECJUK increased the intracellular reduced glutathione level and suppressed 12-O-tetradecanoylphorbol acetate-induced 8-hydroxyguanosine formation without affecting the overall viability. Collectively, our results provide evidence that ECJUK can protect against oxidative stress-mediated damages through the activation of Nrf2/ARE-dependent phase II cytoprotective gene expression. PMID:27051652

  15. T-plastin expression downstream to the calcineurin/NFAT pathway is involved in keratinocyte migration.

    PubMed

    Brun, Cécilia; Demeaux, Agathe; Guaddachi, Frédéric; Jean-Louis, Francette; Oddos, Thierry; Bagot, Martine; Bensussan, Armand; Jauliac, Sébastien; Michel, Laurence

    2014-01-01

    Cutaneous wound healing requires keratinocyte proliferation, migration and differentiation to restore the barrier function of the skin. The calcineurin/nuclear factor of activated-T-cell (NFAT) signaling pathway has been recently shown to be involved in keratinocyte growth, differentiation and migration. It is induced by an increased intracellular calcium rate and its inhibition results in decreased capacities of keratinocytes to migrate. Nevertheless, the link between calcineurin activation and keratinocyte migration remains unknown. Recently, Orai1, a pore subunit of a store-operated calcium channel that favors calcium influx, was shown to play a critical role to control proliferation and migration of basal keratinocytes. Of interest, the actin-bundling T-plastin is crucial in cell motility through cross-linking to actin filament and its synthesis was shown to be induced by calcium influx and regulated by the calcineurin/NFAT pathway in tumor Sezary cells. We investigated herein the role of the calcineurin/NFAT pathway-dependent T-plastin in keratinocyte migration, by quantifying T-plastin expression in keratinocytes and by analyzing their migration under calcineurin inhibition or knockdown of NFAT2 or T-plastin. We did confirm the role of the calcineurin/NFAT pathway in keratinocyte migration as shown by their decreased capacities to migrate after FK506 treatment or siNFAT2 transfection in both scratching and Boyden assays. The expression of NFAT2 and T-plastin in keratinocytes was decreased under FK506 treatment, suggesting that T-plastin plays a role in keratinocyte migration downstream to the calcineurin/NFAT pathway. Accordingly, siRNA knockdown of T-plastin expression also decreased their migration capacities. Actin lamellipodia formation as well as FAK and β6-integrin expression were also significantly decreased after treatment with FK506 or siRNA, reinforcing that NFAT2-dependent T-plastin expression plays a role in keratinocyte migration. These results

  16. Cyclic stretch induces upregulation of endothelin-1 with keratinocytes in vitro: Possible role in mechanical stress-induced hyperpigmentation

    SciTech Connect

    Kurita, Masakazu; Okazaki, Mutsumi; Fujino, Takashi; Takushima, Akihiko; Harii, Kiyonori

    2011-05-27

    Highlights: {yields} Influence of cyclic stretch on melanogenetic paracrine cytokines was investigated. {yields} Keratinocyte-derived endothelin-1 was upregulated with cyclic stretch. {yields} Degree of upregulation increases dose-dependently. {yields} This upregulation possibly plays a role in the pathogenesis of pigmented disorders. -- Abstract: The aim of this study was to investigate the possible pathological relation between mechanical stress and hyperpigmentation. We did this by investigating the influence of cyclic stretch on the expression of keratinocyte- and fibroblast-derived melanogenetic paracrine cytokines in vitro. Using primary human keratinocytes and fibroblasts, alterations of mRNA expression of melanogenetic paracrine cytokines due to cyclic stretch were investigated using a real-time polymerase chain reaction (PCR). The cytokines included basic fibroblast growth factor (bFGF), stem cell factor (SCF), granulocyte/macrophage colony-stimulating factor, interleukin-1{alpha}, and endothelin-1 (ET-1) for keratinocytes and bFGF, SCF, and hepatocyte growth factor for fibroblasts. The dose dependence of keratinocyte-derived ET-1 upregulation was further investigated using real-time PCR and an enzyme-linked immunosorbent assay. We also investigated the effects of cyclic stretch on the proliferation and differentiation of keratinocytes. Among the melanogenetic paracrine cytokines investigated, keratinocyte-derived ET-1 was consistently upregulated in all four cell lines. The degree of upregulation increased with the degree of the length and frequency of the stretch; in contrast, cell number and differentiation markers showed no obvious alterations with cyclic stretch. Keratinocyte-derived ET-1 upregulation possibly plays a significant role in the pathogenesis of pigmented disorders, such as friction melanosis, caused by mechanical stress.

  17. Effect of various metals on intercellular adhesion molecule-1 expression and tumour necrosis factor alpha production by normal human keratinocytes.

    PubMed

    Guéniche, A; Viac, J; Lizard, G; Charveron, M; Schmitt, D

    1994-01-01

    Nickel, cobalt and chromium are metals very often implicated in allergic contact dermatitis. In vivo, keratinocytes, which are the first target cells, can be directly activated to participate in the local reaction, especially through the expression of the membrane antigen ICAM-1, a ligand of the leucocyte antigen LFA-1, and the production of cytokines. Our aim was to assess the effects of sensitizing metal haptens (nickel, cobalt and chromium) compared with the toxic metal cadmium on the induction of ICAM-1 and the production of TNF alpha by epidermal cells. For this purpose, normal human keratinocytes obtained during plastic skin surgery were cultured in low-calcium defined medium (MCDB153) and the metals were used in non-toxic concentrations. Using FACS analysis, ICAM-1 expression was found to be induced only by nickel. This stimulation appeared as early as 24 h after stimulation. All the metals induced a low expression of TNF alpha detectable by immunocytochemistry correlating with the induction of the nuclear stress protein Hsp72 which is closely linked genetically with the TNF alpha locus. However, only Ni2+, Co2+ and Cr2+ induced a significant release of TNF alpha detectable by ELISA after 48 h stimulation. This secretion was lower than that observed with known stimulants such as lipopolysaccharide. These results indicate that the metals studied are able to induce an aggressive cellular effect, and that nickel, by its ICAM-1 induction, may play a major role in the keratinocyte activation state during allergic contact dermatitis. PMID:7864660

  18. Co-immobilization of gradient-patterned growth factors for directed cell migration

    PubMed Central

    Stefonek-Puccinelli, Tracy Jane; Masters, Kristyn S.

    2009-01-01

    Cell migration is critically important for the repair of chronic wounds, which cost billions of dollars each year to treat and can lead to serious complications, including amputation and death. Growth factors, including epidermal growth factor (EGF) and insulin-like growth factor (IGF-1), are known to be deficient in chronic wounds; unfortunately, traditional delivery of soluble growth factors to wounds is expensive and complicated by their degradation. We have previously shown that directed and accelerated keratinocyte migration could be achieved by creating immobilized gradients of EGF. In this work, we have optimized EGF gradients for cell migration, synthesized and characterized gradient patterns of IGF-1, and tested for migration synergy upon combination of EGF and IGF-1 patterns. An optimal EGF concentration and pattern were identified, resulting in migration that was almost 10-fold that achieved on unpatterned controls. Immobilization of IGF-1 gradients also accelerated and directed keratinocyte migration (p<0.05), however, no difference in migration was found across various IGF-1 concentrations or gradient patterns. Although combining EGF with IGF-1 patterns did not accelerate migration beyond levels achieved using EGF alone, these methods can be applied to create other types of multi-component gradients that will ultimately be utilized to create 3-D bioactive wound dressings. PMID:18850272

  19. Fetal fibroblasts and keratinocytes with immunosuppressive properties for allogeneic cell-based wound therapy.

    PubMed

    Zuliani, Thomas; Saiagh, Soraya; Knol, Anne-Chantal; Esbelin, Julie; Dréno, Brigitte

    2013-01-01

    Fetal skin heals rapidly without scar formation early in gestation, conferring to fetal skin cells a high and unique potential for tissue regeneration and scar management. In this study, we investigated the possibility of using fetal fibroblasts and keratinocytes to stimulate wound repair and regeneration for further allogeneic cell-based therapy development. From a single fetal skin sample, two clinical batches of keratinocytes and fibroblasts were manufactured and characterized. Tolerogenic properties of the fetal cells were investigated by allogeneic PBMC proliferation tests. In addition, the potential advantage of fibroblasts/keratinocytes co-application for wound healing stimulation has been examined in co-culture experiments with in vitro scratch assays and a multiplex cytokines array system. Based on keratin 14 and prolyl-4-hydroxylase expression analyses, purity of both clinical batches was found to be above 98% and neither melanocytes nor Langerhans cells could be detected. Both cell types demonstrated strong immunosuppressive properties as shown by the dramatic decrease in allogeneic PBMC proliferation when co-cultured with fibroblasts and/or keratinocytes. We further showed that the indoleamine 2,3 dioxygenase (IDO) activity is required for the immunoregulatory activity of fetal skin cells. Co-cultures experiments have also revealed that fibroblasts-keratinocytes interactions strongly enhanced fetal cells secretion of HGF, GM-CSF, IL-8 and to a lesser extent VEGF-A. Accordingly, in the in vitro scratch assays the fetal fibroblasts and keratinocytes co-culture accelerated the scratch closure compared to fibroblast or keratinocyte mono-cultures. In conclusion, our data suggest that the combination of fetal keratinocytes and fibroblasts could be of particular interest for the development of a new allogeneic skin substitute with immunomodulatory activity, acting as a reservoir for wound healing growth factors.

  20. Development of an inducible gene expression system for primary murine keratinocytes

    PubMed Central

    Nagarajan, Priyadharsini

    2008-01-01

    Background The tetracycline (Tet) responsive system is a valuable tool that is routinely used in a wide variety of mammalian cells for regulatable expression of gene products. However, technical difficulties such as harsh selection conditions and extensive screening processes to identify suitably responsive clones limit the generation of stable cell lines. Hence, application of this system in mammalian cells with relatively slow growth rates and / or the capacity to undergo terminal differentiation such as primary mouse keratinocytes is particularly challenging. Objective To our knowledge, no Tet-responsive stable cell lines have been generated from mouse keratinocytes, presumably due to their sensitivity to selection conditions. Our goal was to utilize a modified and robust Tet-expression system to generate a stable primary mouse keratinocyte cell line. These cells could be then utilized for conditional expression of potentially toxic proteins in an inducible fashion. Methods We utilized a eukaryotic promoter instead of a viral promoter to express a modified reverse tetracycline transactivator in mouse keratinocytes and optimized the selection process for generating stable cell lines. Results Here, we report the generation of a stable mouse keratinocyte cell line for Tet-regulated gene expression with minimal leakiness and high degree of Tet responsivity. This mouse keratinocyte cell line was further engineered for generation of a double stable cell line, which expresses the transcription factor AP-2α in an inducible manner. Importantly, the selected cells retain their inherent keratinocyte morphology, respond to differentiation signals and exhibit a persistent and highly tunable Tet inducibility upon continuous culturing. Conclusion We have generated a tetracycline inducible gene expression model system in mouse epidermal keratinocytes. Such inducible cell lines will serve as valuable in vitro models for future gain-of-function and loss-of-function studies. PMID

  1. Growth factors in orthopedic surgery

    PubMed Central

    Zaharia, C; Despa, N; Simionescu, M; Jinga, V; Fleseriu, I

    2010-01-01

    Growth factors have represented an essential issue of interest for the researchers and clinicians in orthopedics and trauma over the last 40 years. In the last 10 to 15 years, the advances registered in this field have permitted the identification of the most active cellular and humoral factors as well as the improvement of their use in the orthopedic and trauma surgery. Their domain of application has been continuously enlarged and the results have been visible from the beginning. The authors present their appreciation on the actual state of this subject as well as their experience with results and related conclusions. PMID:20302195

  2. Portulaca oleracea L. aids calcipotriol in reversing keratinocyte differentiation and skin barrier dysfunction in psoriasis through inhibition of the nuclear factor κB signaling pathway

    PubMed Central

    ZHAO, HENGGUANG; LI, SHUANG; LUO, FULING; TAN, QIAN; LI, HUI; ZHOU, WEIKANG

    2015-01-01

    Psoriasis affects 2–4% of the population worldwide and its treatment is currently far from satisfactory. Calcipotriol and Portulaca oleracea have been reported to exhibit the capacity to inhibit inflammation in psoriatic patients and improve their clinical condition. However, the efficacy of a combination regimen of these two components remains unknown. The aim of the present study was to explore the therapeutic efficacy of P. oleracea extract combined with calcipotriol on plaque psoriasis and its potential mechanism. Eleven patients with plaque psoriasis were treated with humectant containing the active ingredients of P. oleracea extract, with or without 0.005% calcipotriol ointment in a right-left bilateral lesion self-control study. Differences were evaluated by investigation of the clinical efficacy, adverse effects, skin barrier function, histological structure, expression and proliferation of keratinocytes, differentiation markers (cytokeratin 10, filaggrin and loricrin), inflammatory factors [tumor necrosis factor (TNF)-α and interleukin (IL)-8], as well as the status of the nuclear factor κB (NF-κB) pathway. The combination of P. oleracea and calcipotriol was revealed to decrease adverse effects, reduce transepidermal water loss, potently reverse keratinocyte differentiation dysfunction, and inhibit the expression of TNF-α and IL-8 and the phosphorylation of the NF-κB inhibitor IκBα. This treatment is therefore anticipated to be suitable for use as a novel adjuvant therapy for psoriatic patients. PMID:25574190

  3. Transforming growth factor-beta 1 and fibroblast growth factors in rat growth plate.

    PubMed

    Jingushi, S; Scully, S P; Joyce, M E; Sugioka, Y; Bolander, M E

    1995-09-01

    Chondrocytes in the growth plate progress in an orderly fashion from resting through proliferating to hypertrophic cells. In the region of hypertrophic chondrocytes, the cartilage is invaded by capillary loops and endochondral ossification is initiated. It is currently believed that growth factors may regulate the proliferation and maturation of chondrocytes and the synthesis of extracellular matrix in the growth plate. The ordered sequence of proliferation and differentiation observed in the growth plate provides a unique opportunity to study the role of acidic fibroblast growth factor, basic fibroblast growth factor, and transforming growth factor-beta 1 in the regulation of these processes. In this study, expression of the mRNA of these growth factors was examined using total RNA extracted from the physis and epiphysis of rat tibias. Transforming growth factor-beta 1 mRNA was detected by Northern hybridization. Expression of the genes encoding acidic and basic fibroblast growth factors was demonstrated by polymerase chain reaction amplification. In addition, using polyclonal antibodies against these growth factors, we localized them by immunohistochemical analysis. Strong intracellular staining with a predominantly nuclear pattern was observed in chondrocytes from the proliferating and upper hypertrophic zones. In contrast, chondrocytes in the resting zone stained only faintly for the presence of these growth factors. Some chondrocytes in the resting zone adjacent to the proliferating zone stained with these antibodies, and the antibodies also stained cells in the zone of Ranvier, which regulates latitudinal bone growth. Lastly, the location of transforming growth factor-beta 1 was examined further with use of a polyclonal antipeptide antibody specific for its extracellular epitope.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7472755

  4. Sgk3 links growth factor signaling to maintenance of progenitor cells in the hair follicle.

    PubMed

    Alonso, Laura; Okada, Hitoshi; Pasolli, Hilda Amalia; Wakeham, Andrew; You-Ten, Annick Itie; Mak, Tak W; Fuchs, Elaine

    2005-08-15

    Tyrosine kinase growth factor receptor signaling influences proliferation, survival, and apoptosis. Hair follicles undergo cycles of proliferation and apoptotic regression, offering an excellent paradigm to study how this transition is governed. Several factors are known to affect the hair cycle, but it remains a mystery whether Akt kinases that are downstream of growth factor signaling impact this equilibrium. We now show that an Akt relative, Sgk (serum and glucocorticoid responsive kinase) 3, plays a critical role in this process. Hair follicles of mice lacking Sgk3 fail to mature normally. Proliferation is reduced, apoptosis is increased, and follicles prematurely regress. Maintenance of the pool of transiently amplifying matrix cells is impaired. Intriguingly, loss of Sgk3 resembles the gain of function of epidermal growth factor signaling. Using cultured primary keratinocytes, we find that Sgk3 functions by negatively regulating phosphatidylinositol 3 kinase signaling. Our results reveal a novel and important function for Sgk3 in controlling life and death in the hair follicle.

  5. A comparative study of leukaemia inhibitory factor and interleukin-1alpha intracellular content in a human keratinocyte cell line after exposure to cosmetic fragrances and sodium dodecyl sulphate.

    PubMed

    Parodi, Alessandro; Sanguineti, Roberta; Catalano, Mariafrancesca; Penco, Susanna; Pronzato, Maria Adelaide; Scanarotti, Chiara; Bassi, Anna Maria

    2010-02-01

    According to European laws animal testing in cosmetic industry will be prohibited in a few years and it will be replaced by alternative methods based on cell and tissue culture. Many ingredients of cosmetic formulations are potentially causes of skin inflammation and sensibilization. Since cytotoxicity is known, among other factors, to trigger irritation, in an alternative model for evaluation of skin irritation, it can be considered also the precocious release of inflammatory mediators, i.e. cytokines, originating mainly from keratinocytes. In this in vitro study we have analysed some parameters directly or indirectly related to irritation/inflammation, in NCTC 2544 human keratinocytes during short-time exposure to some potential irritants cosmetic fragrances, included in the European Laws 2003/15/EEC. IIC50 was extrapolated by MTT and NRU viability indexes after exposure of cell ultures to Geraniol Limonene and Benzylic Alcohol for 1, 3 and 6h. NCTC cells were then exposed to sub-toxic doses of selected compounds and interleukin-1alpha (IL-1alpha) and leukaemia inhibitory factor (LIF) expressions were analysed as early proinflammatory cytokines. To our knowledge our findings demonstrated for the first time that NCTC cells synthesize and modulate LIF after exposure to selected irritating stimuli. Moreover, our results give evidence on LIF role as in vitro precocious endpoint for the assessment of the risk in cosmetic field, because its response under irritation stimuli is very quick and comparable to IL-1alpha.

  6. Epidermal growth factor and growth in vivo

    SciTech Connect

    Rhodes, J.A.

    1986-01-01

    Epidermal growth factor (EGF) causes a dose-dependent thickening of the epidermis in suckling mice. The cellular mechanisms underlying this thickening were analyzed by measuring the effect of EGF on the cell-cycle. Neonatal mice were given daily injections of either 2ug EGF/g body weight/day or an equivalent volume of saline, and on the seventh day received a single injection of /sup 3/H-thymidine. At various times the mice were perfused with fixative; 1um sections of skin were stained with a modification of Harris' hematoxylin and were autoradiographed. The sections were analyzed using three methods based on the dependence on time after injection of /sup 3/H-thymidine of: frequency of labelled mitoses, labelling index, and reciprocal grains/nucleus. It was found that EGF caused a two-fold increase in the cell production rate. The effect of exogenous EGF on the morphology of gastric mucosa and incisors of suckling mice was also studied. The gastric mucosa appeared thicker in EGF-treated animals, but the effect was not statistically significant. In contrast to its effect on epidermis and gastric mucosa, EGF caused a significant, dose-dependent decrease in the size of the incisors. Because the mouse submandibular salivary gland is the major source of EGF the effect of sialoadenectomy on female reproductive functions was examined. Ablation of the submandibular gland had no effect on: length of estrus cycle, ability of the female to produce litters, length of the gestation period, litter size, and weight of the litter at birth. There was also no effect on survival of the offspring or on age at which the eyelids separated.

  7. Keratinocytes from APP/APLP2-deficient mice are impaired in proliferation, adhesion and migration in vitro

    SciTech Connect

    Siemes, Christina; Quast, Thomas; Kummer, Christiane; Wehner, Sven; Kirfel, Gregor; Mueller, Ulrike; Herzog, Volker . E-mail: Herzog@uni-bonn.de

    2006-07-01

    Growing evidence shows that the soluble N-terminal form (sAPP{alpha}) of the amyloid precursor protein (APP) represents an epidermal growth factor fostering keratinocyte proliferation, migration and adhesion. APP is a member of a protein family including the two mammalian amyloid precursor-like proteins APLP1 and APLP2. In the mammalian epidermis, only APP and APLP2 are expressed. APP and APLP2-deficient mice die shortly after birth but do not display a specific epidermal phenotype. In this report, we investigated the epidermis of APP and/or APLP2 knockout mice. Basal keratinocytes showed reduced proliferation in vivo by about 40%. Likewise, isolated keratinocytes exhibited reduced proliferation rates in vitro, which could be completely rescued by either exogenously added recombinant sAPP{alpha}, or by co-culture with dermal fibroblasts derived from APP knockout mice. Moreover, APP-knockout keratinocytes revealed reduced migration velocity resulting from severely compromised cell substrate adhesion. Keratinocytes from double knockout mice died within the first week of culture, indicating essential functions of APP-family members for survival in vitro. Our data indicate that sAPP{alpha} has to be considered as an essential epidermal growth factor which, however, in vivo can be functionally compensated to a certain extent by other growth factors, e.g., factors released from dermal fibroblasts.

  8. Endothelins secreted from human keratinocytes are intrinsic mitogens for human melanocytes.

    PubMed

    Imokawa, G; Yada, Y; Miyagishi, M

    1992-12-01

    We recently demonstrated that human melanocyte proliferation and differentiation could be stimulated by endothelin (ET) derivatives via a receptor-mediated signal transduction pathway (Yada, Y., Higuchi, K., and Imokawa, G. (1991) J. Biol. Chem. 266, 18352-18357). We show here that the growth factors for human melanocytes are produced and secreted by the surrounding cells, namely human keratinocytes for ET-1 and Big-ET-1. Northern blots have revealed the presence of ET-1 gene transcripts in proliferating human keratinocytes. The ET-1 production by human keratinocytes increased after irradiation with ultraviolet B (UVB) in a dose-dependent manner, accompanied by the significant secretion of interleukin 1 alpha (IL-1 alpha). Among the cytokines related to UVB-induced cellular reactions and keratinocyte growth, only IL-1 alpha and -1 beta stimulated the secretion of ET-1 and Big-ET-1 but not of ET-3 and Big-ET-3 in a time-dependent manner. Northern blots for IL-1 alpha-stimulated or UVB-exposed human keratinocytes revealed that production of ET-1 gene transcripts markedly increased (by about 300 or 1,200%) with constant levels of beta-actin gene transcripts. In a parallel study, the medium conditioned by UVB-exposed human keratinocytes elicited a significant anti-ET-1 antibody-suppressible increase in DNA synthesis by cultured human melanocytes in a UV dose-dependent manner, which was associated with a marked and rapid (80 s) increase in the intracellular calcium level upon incubation with human melanocytes. These studies suggest that ETs produced and secreted by keratinocytes play an essential role in the maintenance of melanocyte proliferation and UV hyperpigmentation in the epidermis.

  9. Human papillomavirus causes an angiogenic switch in keratinocytes which is sufficient to alter endothelial cell behavior

    SciTech Connect

    Chen, W.; Li, F.; Mead, L.; White, H.; Walker, J.; Ingram, D.A.; Roman, A.

    2007-10-10

    One of the requirements for tumor growth is the ability to recruit a blood supply, a process known as angiogenesis. Angiogenesis begins early in the progression of cervical disease from mild to severe dysplasia and on to invasive cancer. We have previously reported that expression of human papillomavirus type 16 E6 and E7 (HPV16 E6E7) proteins in primary foreskin keratinocytes (HFKs) decreases expression of two inhibitors and increases expression of two angiogenic inducers [Toussaint-Smith, E., Donner, D.B., Roman, A., 2004. Expression of human papillomavirus type 16 E6 and E7 oncoproteins in primary foreskin keratinocytes is sufficient to alter the expression of angiogenic factors. Oncogene 23, 2988-2995]. Here we report that HPV-induced early changes in the keratinocyte phenotype are sufficient to alter endothelial cell behavior both in vitro and in vivo. Conditioned media from HPV16 E6E7 expressing HFKs as well as from human cervical keratinocytes containing the intact HPV16 were able to stimulate proliferation and migration of human microvascular endothelial cells. In addition, introduction of the conditioned media into immunocompetent mice using a Matrigel plug model resulted in a clear angiogenic response. These novel data support the hypothesis that HPV proteins contribute not only to the uncontrolled keratinocyte growth seen following HPV infection but also to the angiogenic response needed for tumor formation.

  10. Experience gained during the long term cultivation of keratinocytes for treatment of burns patients.

    PubMed

    Dragúňová, Jana; Kabát, Peter; Koller, Ján; Jarabinská, Valéria

    2012-08-01

    Both allogenic and autologous cultured skin cells have been used clinically on burn patients. In vitro cultivation of human keratinocytes has been routinely provided by the Central Tissue Bank in Bratislava since 1996, with an average annual production of around 7,000 cm(2). Keratinocytes have been cultivated using a version of the original by Rheinwald and Green (Cell 6:317-330, 1975) methodology which has been modified over time in our laboratory as we gained more experience with this serial passage system. We have observed that the growth of cultured keratinocytes depends on several important factors, including the timing of skin sample procurement, the method of skin sample procurement, the general condition of the patient, the quality and composition of the culture media and, to a lesser extent, the age of the patient. We aim to share our experience with other cell cultivation facilities. PMID:21847560

  11. RIP2: A novel player in the regulation of keratinocyte proliferation and cutaneous wound repair?

    SciTech Connect

    Adams, Stephanie; Valchanova, Ralitsa S.; Munz, Barbara

    2010-03-10

    We could recently demonstrate an important role of receptor interacting protein 4 (RIP4) in the regulation of keratinocyte differentiation. Now, we analyzed a potential role of the RIP4 homolog RIP2 in keratinocytes. Specifically, we demonstrate here that rip2 expression is induced by scratch-wounding and after the induction of differentiation in these cells. Furthermore, serum growth factors and cytokines can induce rip2, with TNF-{alpha}-dependent induction being dependent on p38 MAPK. In addition, we demonstrate that scratch-induced upregulation of rip2 expression is completely blocked by the steroid dexamethasone. Since we also show that RIP2 is an important player in the regulation of keratinocyte proliferation, these data suggest that inhibition of rip2 upregulation after wounding might contribute to the reduced and delayed wound re-epithelialization phenotype seen in glucocorticoid-treated patients.

  12. Arsenite-mediated promotion of anchorage-independent growth of HaCaT cells through placental growth factor.

    PubMed

    Yajima, Ichiro; Kumasaka, Mayuko Y; Ohnuma, Shoko; Ohgami, Nobutaka; Naito, Hisao; Shekhar, Hossain U; Omata, Yasuhiro; Kato, Masashi

    2015-04-01

    Various cancers including skin cancer are increasing in 45 million people exposed to arsenic above the World Health Organization's guideline value of 10 μg l(-1). However, there is limited information on key molecules regulating arsenic-mediated carcinogenesis. Our fieldwork in Bangladesh demonstrated that levels of placental growth factor (PlGF) in urine samples from residents of cancer-prone areas with arsenic-polluted drinking water were higher than those in urine samples from residents of an area that was not polluted with arsenic. Our experimental study in human nontumorigenic HaCaT skin keratinocytes showed that arsenite promoted anchorage-independent growth with increased expression and secretion of PlGF, a ligand of vascular endothelial growth factor receptor1 (VEGFR1), and increased VEGFR1/mitogen-activated protein kinase/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) activities. The arsenite-mediated promotion of anchorage-independent growth was strongly inhibited by PlGF depletion with decreased activities of the PlGF/VEGFR1/MEK/ERK pathway. Moreover, arsenite proteasome-dependently degrades metal-regulatory transcription factor-1 (MTF-1) protein, resulting in a decreased amount of MTF-1 protein binding to the PlGF promoter. MTF-1 negatively controlled PlGF transcription in HaCaT cells, resulting in increased PlGF transcription. These results suggest that arsenite-mediated MTF-1 degradation enhances the activity of PlGF/VEGFR1/MEK/ERK signaling, resulting in promotion of the malignant transformation of keratinocytes. Thus, this study proposed a molecular mechanism for arsenite-mediated development of skin cancer. PMID:25493652

  13. Arsenite-mediated promotion of anchorage-independent growth of HaCaT cells through placental growth factor.

    PubMed

    Yajima, Ichiro; Kumasaka, Mayuko Y; Ohnuma, Shoko; Ohgami, Nobutaka; Naito, Hisao; Shekhar, Hossain U; Omata, Yasuhiro; Kato, Masashi

    2015-04-01

    Various cancers including skin cancer are increasing in 45 million people exposed to arsenic above the World Health Organization's guideline value of 10 μg l(-1). However, there is limited information on key molecules regulating arsenic-mediated carcinogenesis. Our fieldwork in Bangladesh demonstrated that levels of placental growth factor (PlGF) in urine samples from residents of cancer-prone areas with arsenic-polluted drinking water were higher than those in urine samples from residents of an area that was not polluted with arsenic. Our experimental study in human nontumorigenic HaCaT skin keratinocytes showed that arsenite promoted anchorage-independent growth with increased expression and secretion of PlGF, a ligand of vascular endothelial growth factor receptor1 (VEGFR1), and increased VEGFR1/mitogen-activated protein kinase/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) activities. The arsenite-mediated promotion of anchorage-independent growth was strongly inhibited by PlGF depletion with decreased activities of the PlGF/VEGFR1/MEK/ERK pathway. Moreover, arsenite proteasome-dependently degrades metal-regulatory transcription factor-1 (MTF-1) protein, resulting in a decreased amount of MTF-1 protein binding to the PlGF promoter. MTF-1 negatively controlled PlGF transcription in HaCaT cells, resulting in increased PlGF transcription. These results suggest that arsenite-mediated MTF-1 degradation enhances the activity of PlGF/VEGFR1/MEK/ERK signaling, resulting in promotion of the malignant transformation of keratinocytes. Thus, this study proposed a molecular mechanism for arsenite-mediated development of skin cancer.

  14. Antithrombin Regulates Matriptase Activity Involved in Plasmin Generation, Syndecan Shedding, and HGF Activation in Keratinocytes

    PubMed Central

    Chen, Ya-Wen; Xu, Zhenghong; Baksh, Adrienne N. H.; Wang, Jehng-Kang; Chen, Chiu-Yuan; Swanson, Richard; Olson, Steve T.; Kataoka, Hiroaki; Johnson, Michael D.; Lin, Chen-Yong

    2013-01-01

    Matriptase, a membrane-associated serine protease, plays an essential role in epidermal barrier function through activation of the glycosylphosphatidylinositol (GPI)-anchored serine protease prostasin. The matriptase-prostasin proteolytic cascade is tightly regulated by hepatocyte growth factor activator inhibitor (HAI)-1 such that matriptase autoactivation and prostasin activation occur simultaneously and are followed immediately by the inhibition of both enzymes by HAI-1. However, the mechanisms whereby matriptase acts on extracellular substrates remain elusive. Here we report that some active matriptase can escape HAI-1 inhibition by being rapidly shed from the cell surface. In the pericellular environment, shed active matriptase is able to activate hepatocyte growth factor (HGF), accelerate plasminogen activation, and shed syndecan 1. The amount of active matriptase shed is inversely correlated with the amount of antithrombin (AT) bound to the surface of the keratinocytes. Binding of AT to the surface of keratinocytes is dependent on a functional heparin binding site, Lys-125, and that the N-glycosylation site Asn-135 be unglycosylated. This suggests that β-AT, and not α-AT, is responsible for regulation of pericellular matriptase activity in keratinocytes. Keratinocytes appear to rely on AT to regulate the level of pericellular active matriptase much more than breast and prostate epithelial cells in which AT regulation of matriptase activity occurs at much lower levels than keratinocytes. These results suggest that keratinocytes employ two distinct serine protease inhibitors to control the activation and processing of two different sets of matriptase substrates leading to different biological events: 1) HAI-1 for prostasin activation/inhibition, and 2) AT for the pericellular proteolysis involved in HGF activation, accelerating plasminogen activation, and shedding of syndecans. PMID:23675430

  15. Persea americana Mill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts

    PubMed Central

    Ramos-Jerz, Maria del R.; Villanueva, Socorro; Jerz, Gerold; Winterhalter, Peter; Deters, Alexandra M.

    2013-01-01

    Methanolic avocado (Persea americana Mill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Their in vitro influence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fraction M.2 composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore, M.2 increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fraction M.6 increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fraction M.7 contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes. PMID:24371457

  16. Persea americana Mill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts.

    PubMed

    Ramos-Jerz, Maria Del R; Villanueva, Socorro; Jerz, Gerold; Winterhalter, Peter; Deters, Alexandra M

    2013-01-01

    Methanolic avocado (Persea americana Mill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Their in vitro influence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fraction M.2 composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore, M.2 increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fraction M.6 increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fraction M.7 contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes. PMID:24371457

  17. Persea americana Mill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts.

    PubMed

    Ramos-Jerz, Maria Del R; Villanueva, Socorro; Jerz, Gerold; Winterhalter, Peter; Deters, Alexandra M

    2013-01-01

    Methanolic avocado (Persea americana Mill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Their in vitro influence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fraction M.2 composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore, M.2 increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fraction M.6 increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fraction M.7 contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes.

  18. Growth Factors in Proliferative Diabetic Retinopathy

    PubMed Central

    Khan, Zia Ali

    2003-01-01

    Many growth factors are implicated in the pathogenesis of proliferative diabetic retinopathy. Alteration of growth factors and their receptors in diabetes has been shown in both experimental and clinical studies. Sustained hyperglycemia resulting from long-standing diabetes leads to several biochemical abnormalities that consequently result in retinal hypoxia. Retinal oxygenation state regulates various growth factors that promote angiogenesis in order to meet the oxygen demands of the tissue. However, unregulated expression of these growth factors and induction of complex cascades leading to augmentation of other proangiogenic factors, which may not be regulated by tissue oxygenation, leads to uncontrolled retinal neovascularization and blindness in diabetic patients. PMID:14668050

  19. Skin Barrier Defects Caused by Keratinocyte-Specific Deletion of ADAM17 or EGFR Are Based on Highly Similar Proteome and Degradome Alterations.

    PubMed

    Tholen, Stefan; Wolf, Cristina; Mayer, Bettina; Knopf, Julia D; Löffek, Stefanie; Qian, Yawen; Kizhakkedathu, Jayachandran N; Biniossek, Martin L; Franzke, Claus-Werner; Schilling, Oliver

    2016-05-01

    Keratinocyte-specific deletion of ADAM17 in mice impairs terminal differentiation of keratinocytes leading to severe epidermal barrier defects. Mice deficient for ADAM17 in keratinocytes phenocopy mice with a keratinocyte-specific deletion of epidermal growth factor receptor (EGFR), which highlights the role of ADAM17 as a "ligand sheddase" of EGFR ligands. In this study, we aim for the first proteomic/degradomic approach to characterize the disruption of the ADAM17-EGFR signaling axis and its consequences for epidermal barrier formation. Proteomic profiling of the epidermal proteome of mice deficient for either ADAM17 or EGFR in keratinocytes at postnatal days 3 and 10 revealed highly similar protein alterations for ADAM17 and EGFR deficiency. These include massive proteome alterations of structural and regulatory components important for barrier formation such as transglutaminases, involucrin, filaggrin, and filaggrin-2. Cleavage site analysis using terminal amine isotopic labeling of substrates revealed increased proteolytic processing of S100 fused-type proteins including filaggrin-2. Alterations in proteolytic processing are supported by altered abundance of numerous proteases upon keratinocyte-specific Adam17 or Egfr deletion, among them kallikreins, cathepsins, and their inhibitors. This study highlights the essential role of proteolytic processing for maintenance of a functional epidermal barrier. Furthermore, it suggests that most defects in formation of the postnatal epidermal barrier upon keratinocyte-specific ADAM17 deletion are mediated via EGFR.

  20. Synthetic heparin-binding growth factor analogs

    DOEpatents

    Pena, Louis A.; Zamora, Paul; Lin, Xinhua; Glass, John D.

    2007-01-23

    The invention provides synthetic heparin-binding growth factor analogs having at least one peptide chain that binds a heparin-binding growth factor receptor, covalently bound to a hydrophobic linker, which is in turn covalently bound to a non-signaling peptide that includes a heparin-binding domain. The synthetic heparin-binding growth factor analogs are useful as soluble biologics or as surface coatings for medical devices.

  1. Epidermal growth factor receptor not equal to nerve growth factor.

    PubMed

    Williams, L R

    1989-01-01

    I am perplexed by the authors' complete lack of definition of neurotrophic factors. The agents Butcher and Woolf want to blame are neurite promoting factors, not neurotrophic factors. Treatment of Alzheimer's disease with NGF antagonists might instead exacerbate the death of both basal forebrain neurons and their cortical target neurons, accelerating the progress of dementia.

  2. Anti-epidermal growth factor receptor skin toxicity: a matter of topical hydration.

    PubMed

    Ferrari, Daris; Codecà, Carla; Bocci, Barbara; Crepaldi, Francesca; Violati, Martina; Viale, Giulia; Careri, Carmela; Caldiera, Sarah; Bordin, Veronica; Luciani, Andrea; Zonato, Sabrina; Cassinelli, Gabriela; Foa, Paolo

    2016-02-01

    Skin toxicity is a frequent complication of anti-epidermal growth factor receptor therapy, which can be an obstacle in maintaining the dose intensity and may negatively impact on the clinical outcome of cancer patients. Skin lesions depend on the disruption of the keratinocyte development pathways and no treatment is clearly effective in resolving the cutaneous alterations frequently found during anti-epidermal growth factor receptor therapy. Among systemic treatments, oral tetracycline proved to be useful in preventing skin manifestations. We describe the case of a patient affected by metastatic colorectal cancer, for whom a combination of chemotherapy and cetuximab was used as second-line treatment. The patient developed a symptomatic papulopustular skin rash that disappeared completely after a twice-daily application of a hydrating and moisturizing cream, mainly consisting of a mixture of paraffin, silicone compounds, and macrogol. The marked cutaneous amelioration allowed the patient to continue cetuximab without any further symptoms and was associated with a partial radiological response.

  3. Autocrine growth factors and solid tumor malignancy.

    PubMed Central

    Walsh, J. H.; Karnes, W. E.; Cuttitta, F.; Walker, A.

    1991-01-01

    The ability of malignant cells to escape the constraint that normally regulate cell growth and differentiation has been a primary focus of attention for investigators of cancer cell biology. An outcome of this attention has been the discovery that the protein products of oncogenes play a role in the activation of growth signal pathways. A second outcome, possibly related to abnormal oncogene expression, has been the discovery that malignant cells frequently show an ability to regulate their own growth by the release of autocrine growth modulatory substances. Most important, the growth of certain malignant cell types has been shown to depend on autocrine growth circuits. A malignant tumor whose continued growth depends on the release of an autocrine growth factor may be vulnerable to treatment with specific receptor antagonists or immunoneutralizing antibodies designed to break the autocrine circuit. Information is rapidly emerging concerning autocrine growth factors in selected human solid tissue malignancy. Images PMID:1926844

  4. Characterization of human gingival keratinocytes cultured in a serum-free medium.

    PubMed

    Wille, J J; Månsson-Rahemtulla, B; Rahemtulla, F

    1990-01-01

    Primary cultures of keratinocytes were established from gingival tissue explanted on the surface of type I collagen gels and fed a serum-containing medium. Cells could be routinely subcultured for at least five passages in a basal nutrient medium (MCDB 153) containing low calcium (0.1 mM), and supplemented with ethanolamine, phosphoethanolamine, hydrocortisone, insulin, epidermal growth factor and protein of bovine pituitary extract. Cells seeded at low densities doubled exponentially in number every 24-30 h and formed a confluent monolayer within 10-14 days. Phase-contrast light and transmission electron microscopy showed that the keratinocyte cultures had features typical of epithelial cells, including desmosomes and perinuclear tonofilament bundles. Immunofluorescent microscopy showed the presence of specific keratin proteins in basal cells of proliferating cultures. Gel electrophoresis of the insoluble cytosolic proteins of gingival and skin keratinocytes showed several differences. Suspension of dividing gingival keratinocytes in 1.3% methylcellulose medium induced greater than 50% cross-linked envelopes, suggesting the existence of a terminal differentiation pathway in gingival basal cells. Clonal growth experiments showed that both insulin and epidermal growth factor were required for optimal clonal growth. The growth of subcultures was arrested and the unstratified epithelial monolayer induced to form a stratified sheet by replacing the growth medium with basal MCDB 153 medium depleted of growth factors and containing 2 mM calcium. Sheets of stratified gingival epithelium formed on and later released from the dish by enzymatic treatment may be suitable for a variety of experimental and clinical uses.

  5. Growth factor transgenes interactively regulate articular chondrocytes.

    PubMed

    Shi, Shuiliang; Mercer, Scott; Eckert, George J; Trippel, Stephen B

    2013-04-01

    Adult articular chondrocytes lack an effective repair response to correct damage from injury or osteoarthritis. Polypeptide growth factors that stimulate articular chondrocyte proliferation and cartilage matrix synthesis may augment this response. Gene transfer is a promising approach to delivering such factors. Multiple growth factor genes regulate these cell functions, but multiple growth factor gene transfer remains unexplored. We tested the hypothesis that multiple growth factor gene transfer selectively modulates articular chondrocyte proliferation and matrix synthesis. We tested the hypothesis by delivering combinations of the transgenes encoding insulin-like growth factor I (IGF-I), fibroblast growth factor-2 (FGF-2), transforming growth factor beta1 (TGF-β1), bone morphogenetic protein-2 (BMP-2), and bone morphogenetic protien-7 (BMP-7) to articular chondrocytes and measured changes in the production of DNA, glycosaminoglycan, and collagen. The transgenes differentially regulated all these chondrocyte activities. In concert, the transgenes interacted to generate widely divergent responses from the cells. These interactions ranged from inhibitory to synergistic. The transgene pair encoding IGF-I and FGF-2 maximized cell proliferation. The three-transgene group encoding IGF-I, BMP-2, and BMP-7 maximized matrix production and also optimized the balance between cell proliferation and matrix production. These data demonstrate an approach to articular chondrocyte regulation that may be tailored to stimulate specific cell functions, and suggest that certain growth factor gene combinations have potential value for cell-based articular cartilage repair.

  6. The epidermal growth factor receptor decreases Stathmin 1 and triggers catagen entry in the mouse.

    PubMed

    Bichsel, Kyle J; Hammiller, Brianna; Trempus, Carol S; Li, Yanhua; Hansen, Laura A

    2016-04-01

    The epidermal growth factor receptor (EGFR) is necessary for normal involution of hair follicles after the growth phase of anagen, although the mechanisms through which it acts are not well understood. In this report, we used transcriptional profiling of microdissected hair follicles from mice with skin-targeted deletion of Egfr to investigate how EGFR activation triggers catagen. Immunofluorescence for phospho-EGFR in mouse skin revealed increased activation of EGFR in follicular keratinocytes at catagen onset. Consistent with other models of EGFR deficiency, mice with skin-targeted deletion of Egfr (Krt14-Cre(+) /Egfr(fl/fl) ) exhibited a delayed and asynchronous catagen entry. Transcriptional profiling at the time of normal catagen onset at post-natal day (P) 17 revealed increased expression of the mitotic regulator Rcc2 in hair follicles lacking EGFR. Rcc2 protein was strongly immunopositive in the nuclei of control follicular keratinocytes at P16 then rapidly decreased until it was undetectable between P18 and 21. In contrast, Rcc2 expression continued in Egfr mutant follicles throughout this period. Proliferation, measured by bromodeoxyuridine incorporation, was also significantly increased in Egfr mutant follicular keratinocytes compared to controls at P18-21. Similarly, Rcc2-regulated mitotic regulator Stathmin 1 was strikingly reduced in control but not Egfr mutant follicles between P17 and P19. Deletion of Stmn1, in turn, accelerated catagen entry associated with premature cessation of proliferation in the hair follicles. These data reveal EGFR suppression of mitotic regulators including Rcc2 and Stathmin 1 as a mechanism for catagen induction in mouse skin.

  7. Growth factor involvement in tension-induced skeletal muscle growth

    NASA Technical Reports Server (NTRS)

    Vandenburgh, Herman H.

    1993-01-01

    Long-term manned space travel will require a better understanding of skeletal muscle atrophy which results from microgravity. Astronaut strength and dexterity must be maintained for normal mission operations and for emergency situations. Although exercise in space slows the rate of muscle loss, it does not prevent it. A biochemical understanding of how gravity/tension/exercise help to maintain muscle size by altering protein synthesis and/or degradation rate should ultimately allow pharmacological intervention to prevent muscle atrophy in microgravity. The overall objective is to examine some of the basic biochemical processes involved in tension-induced muscle growth. With an experimental in vitro system, the role of exogenous and endogenous muscle growth factors in mechanically stimulated muscle growth are examined. Differentiated avian skeletal myofibers can be 'exercised' in tissue culture using a newly developed dynamic mechanical cell stimulator device which simulates different muscle activity patterns. Patterns of mechanical activity which significantly affect muscle growth and metabolic characteristics were found. Both exogenous and endogenous growth factors are essential for tension-induced muscle growth. Exogenous growth factors found in serum, such as insulin, insulin-like growth factors, and steroids, are important regulators of muscle protein turnover rates and mechanically-induced muscle growth. Endogenous growth factors are synthesized and released into the culture medium when muscle cells are mechanically stimulated. At least one family of mechanically induced endogenous factors, the prostaglandins, help to regulate the rates of protein turnover in muscle cells. Endogenously synthesized IGF-1 is another. The interaction of muscle mechanical activity and these growth factors in the regulation of muscle protein turnover rates with our in vitro model system is studied.

  8. Proliferation and motility of HaCaT keratinocyte derivatives is enhanced by fibroblast nemosis

    SciTech Connect

    Raesaenen, Kati; Vaheri, Antti

    2010-06-10

    The role of paracrine tumor-stroma regulation in the progression of cancer is under intense investigation. Activated fibroblasts are key components of the tumor microenvironment providing the soluble factors mediating the regulation. Nemosis is an experimental model to study these parameters: formation of a multicellular spheroid activates fibroblasts and leads to increased production of soluble factors involved in the promotion of growth and motility. Role of nemosis was investigated in the tumorigenesis of HaCaT derivatives representing skin carcinoma progression. Conditioned medium from fibroblast spheroids increased proliferation rate of HaCaT derivatives. Expression of proliferation marker Ki-67 increased significantly in benign A5 and low-grade malignant II-4 cells, but did not further increase in the metastatic RT3 cells. Expression of p63, keratinocyte stem cell marker linked to cancer progression, was augmented by medium from nemotic fibroblasts; this increase was also seen in RT3 cells. Scratch-wound healing of the keratinocytes was enhanced in response to fibroblast nemosis. Neutralizing antibodies against growth factors inhibited wound healing to some extent; the response varied between benign and malignant keratinocytes. Migration and invasion were enhanced by conditioned medium from nemotic fibroblasts in benign and low-grade malignant cells. RT3 keratinocyte migration was further augmented, but invasion was not, indicating their intrinsic capacity to invade. Our data demonstrate that fibroblast nemosis increases proliferation and motility of HaCaT keratinocyte derivatives, and thus nemosis can be used as a model to study the role of soluble factors secreted by fibroblasts in tumor progression.

  9. [Growth factors in proliferative diabetic retinopathy].

    PubMed

    Ioniţă, M

    1997-01-01

    This work presents the possible implications of the angiogenic growth factors and some cell mediators in the initiation and development of the neovascular proliferation in diabetic retinopathy. According to the physiopathologic theories stated above, that are implied in the generation of proliferative diabetic retinopathy, here are some therapeutic experiments based on the action of the angiogenic growth factors. PMID:9409959

  10. Development and bioevaluation of nanofibers with blood-derived growth factors for dermal wound healing.

    PubMed

    Bertoncelj, Valentina; Pelipenko, Jan; Kristl, Julijana; Jeras, Matjaž; Cukjati, Marko; Kocbek, Petra

    2014-09-01

    The aim of our work was to produce a modern nanomaterial with incorporated blood-derived growth factors, produced by electrospinning, applicable in treatment of chronic wounds. Platelet-rich plasma was chosen as a natural source of growth factors. Results showed that platelet-rich plasma stimulates keratinocyte and fibroblast cell growth in vitro. Its optimal concentration in growth medium was 2% (v/v) for both types of skin cells, while higher concentrations caused alterations in cell morphology, with reduced cell mobility and proliferation. In the next step hydrophilic nanofibers loaded with platelet-rich plasma were produced from chitosan and poly(ethylene oxide), using electrospinning. The morphology of nanofibers was stable in aqueous conditions for 72 h. It was shown that electrospinning does not adversely affect the biological activity of platelet-rich plasma. The effects of nanofibers with incorporated platelet-rich plasma on cell proliferation, survival, morphology and mobility were examined. Nanofibers limited cell mobility, changed morphology and stimulated cell proliferation. Despite of the small amount of blood-derived growth factors introduced in cell culture via platelet-rich plasma-loaded nanofibers, such nanofibrillar support significantly induced cell proliferation, indicating synergistic effect of nanotopography and incorporated growth factors. The overall results confirm favorable in vitro properties of produced nanofibers, indicating their high potential as a nanomaterial suitable for delivery of platelet-rich plasma in wound healing applications. PMID:24931341

  11. Growth factors from genes to clinical application

    SciTech Connect

    Sara, V.R. ); Hall, K.; Low, H. )

    1990-01-01

    The last decade has witnessed an explosion in the identification of growth factors and their receptors. This has been greatly facilitated by recombinant DNA technology, which has provided the tools not only to identify these proteins at the gene level but also to produce recombinant proteins for evaluating their biological activities. With the help of such techniques, we are moving toward an understanding of the biosynthesis of growth factors and their receptors, structure-function relationships, as well as mechanisms for intracellular signal transmission. The possibility of modifying these factors has opened new fields of clinical application. In this paper, four major areas of growth factor research are presented: the characterization of growth factor genes and their protein products, growth factor receptors and signal transduction by the receptors to mediate biological action, the biological actions of the various growth factors, and the role of growth factors in health and disease and their possible clinical application. Some of the topics covered include: structure of the IGFs and their variants; isoforms of PDGF receptor types; tyrosine kinase activation; structure of G-proteins in biological membranes; possible therapeutic application of NGF in the treatment of Parkinson's and Alzheimer's diseases; PDGF's possible role in the development of several fibroproliferative diseases and its therapeutic application in wound healing; and the possible use of angiogenic inhibitors in tumor treatment.

  12. growl: Growth factor and growth rate of expanding universes

    NASA Astrophysics Data System (ADS)

    Hamilton, Andrew J. S.

    2015-12-01

    Growl calculates the linear growth factor Da and its logarithmic derivative dln D/dln a in expanding Friedmann-Robertson-Walker universes with arbitrary matter and vacuum densities. It permits rapid and stable numerical evaluation.

  13. EFFECT OF ARSENICALS ON ULTRAVIOLET-RADIATION-INDUCED GROWTH ARREST AND RELATED SIGNALING EVENTS IN HUMAN KERATINOCYTES

    EPA Science Inventory

    The molecular mechanisms mediating arsenic-induced carcinogenesis are not well understood. The role of confounding factors such as ultraviolet radiation (UV), add another level of complexity to the study of arsenic carcinogenesis and the cancer risk assessment to humans. We hypot...

  14. Differential response of nontumorigenic and tumorigenic human papillomavirus type 16-positive epithelial cells to transforming growth factor beta 1.

    PubMed

    Braun, L; Dürst, M; Mikumo, R; Gruppuso, P

    1990-11-15

    The transforming growth factor (TGF) beta s are multifunctional polypeptide growth factors with diverse biological effects, including inhibition of epithelial cell proliferation both in vitro and in vivo. To investigate the possible role of TGF beta 1 in the regulation of papillomavirus infection and papillomavirus-associated transformation, we compared the response to TGF beta 1 of normal keratinocytes, human papillomavirus, type 16 (HPV 16)-positive-immortalized keratinocytes (nontumorigenic), and HPV 16-positive cervical carcinoma cells (tumorigenic) with respect to DNA synthesis and protooncogene expression. All HPV 16-immortalized cell lines were nearly as inhibited by TGF beta 1 as normal keratinocytes, whereas two cervical carcinoma cell lines (Caski and Siha) were refractory to growth inhibition by TGF beta 1. Cell surface receptors for TGF beta 1 were present on both normal and carcinoma cell lines. In all cases, growth inhibition by TGF beta 1 was accompanied by suppression of Steady-state levels of c-myc mRNA. In contrast, TGF beta 1 induced the expression of c-jun mRNA transcripts in normal, immortalized, and tumorigenic cells. We also studied the effect of TGF beta 1 on HPV 16 mRNA expression. Steady-state levels of HPV 16 mRNA transcripts were suppressed by TGF beta 1 in the nontumorigenic HPK cells but were unaffected in the tumorigenic lines. These findings suggest that TGF beta 1 may be an in vivo modulator of HPV infection and that loss of responsiveness to this growth inhibitory signal may be involved in HPV-associated malignant transformation. PMID:2171761

  15. Differential Utilization and Localization of ErbB Receptor Tyrosine Kinases in Skin Compared to Normal and Malignant Keratinocytes1

    PubMed Central

    Stoll, Stefan W; Kansra, Sanjay; Peshick, Scott; Fry, David W; Leopold, Wilbur R; Wiesen, Jane F; Sibilia, Maria; Zhang, Tong; Werb, Zena; Derynck, Rik; Wagner, Erwin F; Elder, James T

    2001-01-01

    Abstract Induction of heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA in mouse skin organ culture was blocked by two pan-ErbB receptor tyrosine kinase (RTK) inhibitors but not by genetic ablation of ErbB1, suggesting involvement of multiple ErbB species in skin physiology. Human skin, cultured normal keratinocytes, and A431 skin carcinoma cells expressed ErbB1, ErbB2, and ErbB3, but not ErbB4. Skin and A431 cells expressed more ErbB3 than did keratinocytes. Despite strong expression of ErbB2 and ErbB3, heregulin was inactive in stimulating tyrosine phosphorylation in A431 cells. In contrast, it was highly active in MDA-MB-453 breast carcinoma cells. ErbB2 displayed punctate cytoplasmic staining in A431 and keratinocytes, compared to strong cell surface staining in MDA-MB-453. In skin, ErbB2 was cytoplasmic in basal keratinocytes, assuming a cell surface pattern in the upper suprabasal layers. In contrast, ErbB1 retained a cell surface distribution in all epidermal layers. Keratinocyte proliferation in culture was found to be ErbB1-RTK-dependent, using a selective inhibitor. These results suggest that in skin keratinocytes, ErbB2 transduces ligand-dependent differentiation signals, whereas ErbB1 transduces ligand-dependent proliferation/survival signals. Intracellular sequestration of ErbB2 may contribute to the malignant phenotype of A431 cells, by allowing them to respond to ErbB1-dependent growth/survival signals, while evading ErbB2-dependent differentiation signals. PMID:11571634

  16. Growth factor involvement in tension-induced skeletal muscle growth

    NASA Technical Reports Server (NTRS)

    Vandenburgh, H. H.

    1987-01-01

    Muscle tissue culture techniques were developed to grow skeletal myofibers which differentiate into more adult-like myofibers. Mechanical simulation studies of these muscle cells in a newly developed mechanical cell simulator can now be performed to study growth processes in skeletal muscle. Conditions in the mechanical cell simulator were defined where mechanical activity can either prevent muscle wasting or stimulate muscle growth. The role of endogenous and exogenous growth factors in tension-induced muscle growth is being investigated under the defined conditions of tissue culture.

  17. Cloning and biological activity of epigen, a novel member of the epidermal growth factor superfamily.

    PubMed

    Strachan, L; Murison, J G; Prestidge, R L; Sleeman, M A; Watson, J D; Kumble, K D

    2001-05-25

    High throughput sequencing of a mouse keratinocyte library was used to identify an expressed sequence tag with homology to the epidermal growth factor (EGF) family of growth factors. We have named the protein encoded by this expressed sequence tag Epigen, for epithelial mitogen. Epigen encodes a protein of 152 amino acids that contains features characteristic of the EGF superfamily. Two hydrophobic regions, corresponding to a putative signal sequence and transmembrane domain, flank a core of amino acids encompassing six cysteine residues and two putative N-linked glycosylation sites. Epigen shows 24-37% identity to members of the EGF superfamily including EGF, transforming growth factor alpha, and Epiregulin. Northern blotting of several adult mouse tissues indicated that Epigen was present in testis, heart, and liver. Recombinant Epigen was synthesized in Escherichia coli and refolded, and its biological activity was compared with that of EGF and transforming growth factor alpha in several assays. In epithelial cells, Epigen stimulated the phosphorylation of c-erbB-1 and mitogen-activated protein kinases and also activated a reporter gene containing enhancer sequences present in the c-fos promoter. Epigen also stimulated the proliferation of HaCaT cells, and this proliferation was blocked by an antibody to the extracellular domain of the receptor tyrosine kinase c-erbB-1. Thus, Epigen is the newest member of the EGF superfamily and, with its ability to promote the growth of epithelial cells, may constitute a novel molecular target for wound-healing therapy. PMID:11278323

  18. Environmental factors influencing growth and pubertal development.

    PubMed Central

    Delemarre-van de Waal, H A

    1993-01-01

    Postnatal growth is based on hereditary signals and environmental factors in a complex regulatory network. Each factor must be in an optimal state for normal growth of the child. Fetal conditions may also have consequences on postnatal height. Intrauterine growth retardation can be recovered postnatally, although postnatal growth remains depressed in about one-third of cases. After birth, the environment may exert either a positive or negative effect on growth. In underdeveloped countries, malnutrition plays a major role in inhibiting the growth process. Children from families of higher socioeconomic classes are taller than their coevals in the lower socioeconomic groups. Urbanization also has a positive effect on growth. Better child care is supported by sufficient food supply, appropriate health and sanitation services, and a higher level of education. Over the last century, these factors have induced a taller stature and a more rapid maturity in Europe, North America, and Australia; a phenomenon which has been referred to as "the secular trend" in growth. Recently, a secular trend has also been reported in some developing countries. Although urbanization in general appears to be associated with better conditions of living, this is not the case in the slums of South America or in Africa where rural children are better off than children living in the poor cities. This paper describes in more detail the different hereditary and environmental factors that act during the fetal period and postnatally, and which play a role in human growth and pubertal development. PMID:8243404

  19. 15-deoxy prostaglandin J2, the nonenzymatic metabolite of prostaglandin D2, induces apoptosis in keratinocytes of human hair follicles: a possible explanation for prostaglandin D2-mediated inhibition of hair growth.

    PubMed

    Joo, Hyun Woo; Kang, Yoo Ri; Kwack, Mi Hee; Sung, Young Kwan

    2016-07-01

    Recent studies have shown that prostaglandin D2 (PGD2) and its nonenzymatic metabolite, 15-deoxy-Δ(12,14)-prostaglandin J2 (15-dPGJ2), inhibit in vitro growth of explanted human hair follicles and inhibit hair growth in mice through the GPR44 (DP2). However, the underlying mechanism is still unclear. In this study, we first investigated the expression of DP2 in human hair follicles and in cultured follicular cells. We found that DP2 is strongly expressed in the outer root sheath (ORS) cells and weakly expressed in the dermal papilla (DP) cells. We observed slight growth stimulation when ORS and DP cells were treated with PGD2. We also observed slight growth stimulation when DP and ORS cells were treated with low concentrations (0.5 and 1 μM) of 15-dPGJ2. However, 5 μM 15-dPGJ2 inhibited the viability and caused apoptosis of both cell types. Exposure of cultured human hair follicles to 15-dPGJ2 resulted in significant apoptosis in follicular keratinocytes. Altogether, our data provide an evidence that 15-dPGJ2 promotes apoptosis in follicular keratinocytes and provide rationale for developing remedies for the prevention and treatment of hair loss based on DP2 antagonism.

  20. Vascular growth factors in neuropsychiatry

    PubMed Central

    Newton, Samuel S.; Fournier, Neil M.; Duman, Ronald S.

    2014-01-01

    Recent advances in understanding the cellular and molecular basis of psychiatric illnesses have shed light on the important role played by trophic factors in modulating functional parameters associated with disease causality and drug action. Disease mechanisms are now thought to involve multiple cell types, including neurons and endothelial cells. These functionally distinct but interactively coupled cell types engage in cellular cross talk via shared and common signaling molecules. Dysregulation in their cellular signaling pathways influences brain function and alters behavioral performance. Multifunctional trophic factors such as VEGF and EPO that possess both neurotrophic and angiogenic actions are of particular interest due to their ability to rescue structural and plasticity deficits in neurons and vasculature. Obtaining insight into the behavioral, cellular and molecular actions of multi-functional trophic factors has the potential to open new and transformative therapeutic approaches. PMID:23475069

  1. Differential control of Notch1 gene transcription by Klf4 and Sp3 transcription factors in normal versus cancer-derived keratinocytes.

    PubMed

    Lambertini, Chiara; Pantano, Serafino; Dotto, G Paolo

    2010-04-28

    In specific cell types like keratinocytes, Notch signaling plays an important pro-differentiation and tumor suppressing function, with down-modulation of the Notch1 gene being associated with cancer development. Besides being controlled by p53, little else is known on regulation of Notch1 gene expression in this context. We report here that transcription of this gene is driven by a TATA-less "sharp peak" promoter and that the minimal functional region of this promoter, which extends from the -342 bp position to the initiation codon, is differentially active in normal versus cancer cells. This GC rich region lacks p53 binding sites, but binds Klf4 and Sp3. This finding is likely to be of biological significance, as Klf4 and, to a lesser extent, Sp3 are up-regulated in a number of cancer cells where Notch1 expression is down-modulated, and Klf4 over-expression in normal cells is sufficient to down-modulate Notch1 gene transcription. The combined knock-down of Klf4 and Sp3 was necessary for the reverse effect of increasing Notch1 transcription, consistent with the two factors exerting an overlapping repressor function through their binding to the Notch1 promoter.

  2. New Clue Found to Growth Factor Action.

    ERIC Educational Resources Information Center

    Hoffman, Michelle

    1991-01-01

    Discussed is the discovery which may help to explain epidermal growth factor effects on the cell skeleton. The role of a protein called profilin in the regulation of the microfilament system is described. (CW)

  3. β4 Integrin and Epidermal Growth Factor Coordinately Regulate Electric Field-mediated Directional Migration via Rac1

    PubMed Central

    Pullar, Christine E.; Baier, Brian S.; Kariya, Yoshinobu; Russell, Alan J.; Horst, Basil A.J.; Marinkovich, M. Peter

    2006-01-01

    Endogenous DC electric fields (EF) are present during embryogenesis and are generated in vivo upon wounding, providing guidance cues for directional cell migration (galvanotaxis) required in these processes. To understand the role of beta (β)4 integrin in directional migration, the migratory paths of either primary human keratinocytes (NHK), β4 integrin-null human keratinocytes (β4−), or those in which β4 integrin was reexpressed (β4+), were tracked during exposure to EFs of physiological magnitude (100 mV/mm). Although the expression of β4 integrin had no effect on the rate of cell movement, it was essential for directional (cathodal) migration in the absence of epidermal growth factor (EGF). The addition of EGF potentiated the directional response, suggesting that at least two distinct but synergistic signaling pathways coordinate galvanotaxis. Expression of either a ligand binding–defective β4 (β4+AD) or β4 with a truncated cytoplasmic tail (β4+CT) resulted in loss of directionality in the absence of EGF, whereas inhibition of Rac1 blinded the cells to the EF even in the presence of EGF. In summary, both the β4 integrin ligand–binding and cytoplasmic domains together with EGF were required for the synergistic activation of a Rac-dependent signaling pathway that was essential for keratinocyte directional migration in response to a galvanotactic stimulus. PMID:16914518

  4. Effects of retinoids on differentiation, lipid metabolism, epidermal growth factor, and low-density lipoprotein binding in squamous carcinoma cells

    SciTech Connect

    Ponec, M.; Weerheim, A. ); Havekes, L. ); Boonstra, J. )

    1987-08-01

    The relationship among keratinocyte differentiation capacity, lipid synthesis, low-density lipoprotein (LDL) metabolism, plasma membrane composition, and epidermal growth factor (EGF) binding has been studied in SCC-12F2 cells. The differentiation capacity of the cells, i.e., ionophore-induced cornified envelope formation, was inhibited by various retinoids and stimulated by hydrocortisone. Retinoids that caused a significant reduction of cornified envelope formation, i.e., retinoic acid and 13-cis-retinoic acid, caused only minor changes in lipid synthesis and plasma membrane composition. Arotinoid ethylsulfone, having a minor effect on cornified envelope formation, caused a drastic inhibition of cholesterol synthesis resulting in changes in the plasma membrane composition. Hydrocortisone stimulated cornified envelope formation but had only minor effects on lipid synthesis and plasma membrane composition. Of all retinoids tested, only arotinoid ethylsulfone caused a drastic increase in EGF binding, while hydrocortisone had no effect. These results clearly demonstrate that the plasma membrane composition is not related to keratinocyte differentiation capacity, but most likely does determine EGF binding. Furthermore, EGF binding does not determine keratinocyte differentiation capacity.

  5. Determination of ligand-binding specificity by alternative splicing: Two distinct growth factor receptors encoded by a single gene

    SciTech Connect

    Miki, T.; Bottaro, D.P.; Fleming, T.P.; Smith, C.L.; Chan, A.M.L.; Aaronson, S.A. ); Burgess, W.H. )

    1992-01-01

    Expression cDNA cloning and structural analysis of the human keratinocyte growth factor receptor (KGFR) revealed identity with one of the fibroblast growth factor (FGF) receptors encoded by the bek gene (FGFR-2), except for a divergent stretch of 49 amino acids in their extracellular domains. Binding assays demonstrated that the KGFR was a high-affinity receptor for both KGF and acidic FGF, while FGFR-2 showed high affinity for basic and acidic FGF but no detectable binding by KGF. Genomic analysis of the bek gene revealed two alternative exons responsible for the region of divergence between the two receptors. The KGFR transcript was specific to epithelial cells, and it appeared to be differentially regulated with respect to the alternative FGFR-2 transcript. Thus, two growth factor receptors with different ligand-binding specificities and expression patterns are encoded by alternative transcripts of the same gene.

  6. Co-culture of melanocytes with adipose-derived stem cells as a potential substitute for co-culture with keratinocytes.

    PubMed

    Kim, Ji-Young; Park, Chan Do; Lee, Joon Ho; Lee, Chang-Hoon; Do, Byung-Rok; Lee, Ai Young

    2012-01-01

    Cell-to-cell interactions between melanocytes and keratinocytes increase the proliferation and migration of melanocytes. In fact, mixed keratinocyte and melanocyte cultures have been used for autologous cell transplantation for treatment of vitiligo. However, this may require taking an amount of skin tissue large enough to leave scars. In this study, the in vitro effect of adipose-derived stem cells (ADSCs) on proliferation, differentiation and migration of melanocytes was compared with that of keratinocytes using immunohistochemistry and a Boyden chamber migration assay. The proliferation and migration of melanocytes was significantly stimulated by co-culture with ADSCs compared with melanocyte monocultures, al-though the effect of ADSCs was less powerful than that of keratinocytes. This may be related to increases in stem cell factor and basic fibroblast growth factor, growth factors for melanocytes, produced by the ADSCs. The ratios of melanocytes stained with antibodies against Trp-2, E-cadherin and N-cadherin were significantly increased by co-culturing with ADSCs compared with co-culturing with keratinocytes as well as melanocyte monocultures. The proportion of less-pigmented melanocytes was also increased and sustained for a longer duration in the presence of ADSCs. Our data show that co-culturing with ADSCs results in increased melanocyte proliferation and migration while reducing differentiation, and could provide a means to treat disorders such as vitiligo.

  7. Expression profiling of cancer-related genes in human keratinocytes following non-lethal ultraviolet B irradiation.

    PubMed

    Murakami, T; Fujimoto, M; Ohtsuki, M; Nakagawa, H

    2001-10-01

    Ultraviolet B irradiation initiates and promotes skin cancers, photo-aging, and immune suppression. In order to elucidate the effect of these processes at the level of gene expression, we used cDNA microarray technology to examine the effect of ultraviolet B irradiation on 588 cancer-related genes in human keratinocytes at 1, 6, and 24 h post-irradiation with a mildly cytotoxic dose of ultraviolet B (170 mJ/cm(2)). The viability of the irradiated keratinocytes was 75% at 24 h post-irradiation. Various cytokeratins and transcription factors were up-regulated within 1 h post-irradiation. After 6 h, expression of a variety of genes related to growth regulation (e.g. p21(WAF1), notch 4, and smoothened), apoptosis (e.g. caspase 10, hTRIP, and CRAF1), DNA repair (ERCC1, XRCC1), cytokines (e.g. IL-6, IL-13, TGF-beta, and endothelin 2), and cell adhesion (e.g. RhoE, and RhoGDI) were altered in human keratinocytes. These data suggest the changes in a cascade of gene expression in human keratinocytes occurring within 24 h after UVB exposure. Although the roles of these cellular genes after UVB-irradiation remain to be elucidated, microarray analysis may provide a new view of gene expression in epidermal keratinocytes following UVB exposure.

  8. Histone H3K27 Demethylase JMJD3 in Cooperation with NF-κB Regulates Keratinocyte Wound Healing.

    PubMed

    Na, Jungtae; Lee, Kwanghyun; Na, Wonho; Shin, Jee-Yoon; Lee, Min-Jung; Yune, Tae Young; Lee, Hae Kwang; Jung, Han-Sung; Kim, Won Sun; Ju, Bong-Gun

    2016-04-01

    Histone H3K27me3 demethylase JMJD3 has been shown to be involved in keratinocyte differentiation and wound healing. However, the exact molecular mechanism underlying JMJD3-mediated keratinocyte wound healing has not been fully elucidated. In this study, we report on the biological function of JMJD3 in keratinocyte wound healing using in vitro cell and in vivo animal models. Our results indicate that JMJD3 up-regulation and NF-κB activation occur in the region of the wound edge during keratinocyte wound healing. We also found that JMJD3 interacts with NF-κB, resulting in increased expression of the inflammatory, matrix metalloproteinase, and growth factor genes via demethylation of H3K27me3 at the gene promoters. Consistently, inactivation of JMJD3 or NF-κB resulted in aberrant keratinocyte wound healing. Our study suggests that regulation of JMJD3 may provide a new therapeutic intervention for treating the chronic skin wound.

  9. Evaluation of emulsion electrospun polycaprolactone/hyaluronan/epidermal growth factor nanofibrous scaffolds for wound healing.

    PubMed

    Wang, Zhenbei; Qian, Yuna; Li, Linhao; Pan, Lianhong; Njunge, Lucy W; Dong, Lili; Yang, Li

    2016-01-01

    Wound healing scaffolds provide cells with structural integrity and can also deliver biological agents to establish a skin tissue-specific microenvironment to regulate cell functions and to accelerate the healing process. In this study, we fabricated biodegradable nanofibrous scaffolds with an emulsion electrospinning technique. The scaffolds were composed of polycaprolactone, hyaluronan and encapsulating epidermal growth factor. The morphology and core-sheath structure of the nanofibers were characterized by scanning electron microscopy and transmission electron microscopy. The scaffolds were also characterized for chemical composition and hydrophilicity with a Fourier-transform infrared analysis, energy dispersive spectroscopy and the water contact angle. An in vitro model protein bovine serum albumin and epidermal growth factor release study was conducted to evaluate the sustained release potential of the core-sheath structured nanofibers with and without the hyaluronan component. Additionally, an in vitro cultivation of human skin keratinocytes (HaCaT) and fibroblasts on polycaprolactone/hyaluronan and polycaprolactone/hyaluronan-epidermal growth factor scaffolds showed a significant synergistic effect of hyaluronan and epidermal growth factor on cell proliferation and infiltration. Furthermore, there was an up-regulation of the wound-healing-related genes collagen I, collagen III and TGF-β in polycaprolactone/hyaluronan/epidermal growth factor scaffolds compared with control groups. In the full-thickness wound model, the enhanced regeneration of fully functional skin was facilitated by epidermal regeneration in the polycaprolactone/hyaluronan/epidermal growth factor treatment group. Our findings suggest that bioactivity and hemostasis of the hyaluronan-based nanofibrous scaffolds have the capability to encapsulate and control the release of growth factors that can serve as skin tissue engineering scaffolds for wound healing.

  10. Use of allogenic epidermal sheets for difficult wound healing: selection and testing of relevant growth factors.

    PubMed

    Auxenfans, C; Colloud, M; Debard, A L; Braye, F M; Amini, M; Allombert-Blaise, V; Builles, N; Claudy, A; Damour, O

    2006-01-01

    The clinical interest of using allogenic epidermal sheets (AES) has largely been shown [1,2,3]. As well as covering, they also stimulate healing, by simultaneously secreting numerous growth factors (GFs), although little is known on their mechanism of action. Our objectives were to: (a) devise a test for the efficacy of AES release, (b) select keratinocyte-secreting strains and optimal culture conditions. Three GFs were selected: IL-1alpha, IL-8 and VEGF. Three different keratinocyte strains were cultured for 3 and 6 days after confluence for 3 passages. Assays were performed after 3 h and 24 h+3 h after dispase treatment (AES conservation for 24 h then change of medium and sampling after 3 h). AES were found to secrete GFs in DMEM and the amounts were greater when cultured for 6 rather than 3 days after confluence. Each strain had different secretory patterns depending on passage and time in culture, this variability being explained by inter-individual heterogeneity.

  11. Identification of copper/zinc superoxide dismutase as a nitric oxide-regulated gene in human (HaCaT) keratinocytes: implications for keratinocyte proliferation.

    PubMed

    Frank, S; Kämpfer, H; Podda, M; Kaufmann, R; Pfeilschifter, J

    2000-03-15

    Recent studies have demonstrated an induction of expression of inducible nitric oxide synthase that is associated with several inflammatory diseases of the skin. To define the mechanisms of action of nitric oxide (NO) in the skin, we attempted to identify genes that are regulated by NO in keratinocytes. Using the human keratinocyte cell line HaCaT as a model system, we identified a Cu/Zn superoxide dismutase (SOD) that was strongly induced by high concentrations (500 microM) of NO-donating agents ¿S-nitrosoglutathione, sodium nitroprusside and (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1,2 -diolate (DETA-NO)¿, but not by serum or by single recombinant growth factors and inflammatory cytokines or by treatment with superoxide anions. Furthermore, endogenously produced NO increased the expression of Cu/Zn SOD mRNA in keratinocytes. Moreover, treatment of HaCaT cells with NO was associated with a biphasic effect on cell proliferation, because low doses (100 microM) of different NO donors (S-nitrosoglutathione and DETA-NO) mediated a proliferative signal to the cells, whereas high concentrations (500 microM) were cytostatic. To determine a possible correlation between the close regulation of Cu/Zn SOD expression and proliferation by NO in keratinocytes, we established a cell line (psp1CZ1N) carrying a human Cu/Zn SOD cDNA under the control of a ponasterone-inducible promoter construct. Ponasterone-induced overexpression of Cu/Zn SOD caused a cytostatic effect in proliferating psp1CZ1N cells. We therefore suggest that the up-regulation of Cu/Zn SOD expression by NO establishes an inhibitory mechanism on keratinocyte proliferation. PMID:10698699

  12. Predictive factors for intrauterine growth restriction

    PubMed Central

    Albu, AR; Anca, AF; Horhoianu, VV; Horhoianu, IA

    2014-01-01

    Abstract Reduced fetal growth is seen in about 10% of the pregnancies but only a minority has a pathological background and is known as intrauterine growth restriction or fetal growth restriction (IUGR / FGR). Increased fetal and neonatal mortality and morbidity as well as adult pathologic conditions are often associated to IUGR. Risk factors for IUGR are easy to assess but have poor predictive value. For the diagnostic purpose, biochemical serum markers, ultrasound and Doppler study of uterine and spiral arteries, placental volume and vascularization, first trimester growth pattern are object of assessment today. Modern evaluations propose combined algorithms using these strategies, all with the goal of a better prediction of risk pregnancies. Abbreviations: SGA = small for gestational age; IUGR = intrauterine growth restriction; FGR = fetal growth restriction; IUFD = intrauterine fetal demise; HIV = human immunodeficiency virus; PAPP-A = pregnancy associated plasmatic protein A; β-hCG = beta human chorionic gonadotropin; MoM = multiple of median; ADAM-12 = A-disintegrin and metalloprotease 12; PP-13 = placental protein 13; VEGF = vascular endothelial growth factor; PlGF = placental growth factor; sFlt-1 = soluble fms-like tyrosine kinase-1; UAD = uterine arteries Doppler ultrasound; RI = resistence index; PI = pulsatility index; VOCAL = Virtual Organ Computer–Aided Analysis software; VI = vascularization index; FI = flow index; VFI = vascularization flow index; PQ = placental quotient PMID:25408721

  13. Insulin-like growth factor 1 and hair growth.

    PubMed

    Su, H Y; Hickford, J G; Bickerstaffe, R; Palmer, B R

    1999-11-01

    Insulin-like growth factor 1 (IGF-1) has been identified as an important growth factor in many biological systems.[1] It shares considerable structural homology with insulin and exerts insulin-like effects on food intake and glucose metabolism. Recently it has been suggested to play a role in regulating cellular proliferation and migration during the development of hair follicles. [2,3] To exert its biological effects, the IGF-1 is required to activate cells by binding to specific cell-surface receptors. The type I IGF receptor (IGF-1R) is the only IGF receptor to have IGF-mediated signaling functions.[1] In circulation, this growth factor mediates endocrine action of growth hormone (GH) on somatic growth and is bound to specific binding proteins (BPs). The latter control IGF transport, efflux from vascular compartments and association with cell surface receptors.[4] In tissues, IGF-1 is produced by mesenchymal type cells and acts in a paracrine and autocrine fashion by binding to the IGF-1R. This binding activates the receptor tyrosine kinase (RTK) that triggers the downstream responses and finally stimulates cell division.[5] IGF-1 may therefore be able to stimulate the proliferation of hair follicle cells through cellular signaling pathways of its receptors. Local infusion of IGF-1 into sheep has been reported to be capable of stimulating protein synthesis in the skin.[6] It may also increase the production of wool keratin. Recently, transgenic mice overexpressing IGF-1 in the skin have been shown to have earlier hair follicle development than controls.[7] In addition, this growth factor plays an important role in many cell types as a survival factor to prevent cell death.[8] This anti-apoptotic function of IGF-1 may be important to the development of follicle cells as follicles undergo a growth cycle where the regressive, catagen phase is apoptosis driven. In this review, the effects of IGF-1 on follicle cell proliferation and differentiation are discussed. In

  14. Organic growth factor requirements of some yeasts.

    PubMed

    Madan, M; Gulati, N

    1980-01-01

    Some sporogenous yeasts (Brettanomyces bruxellensis, Debaryomyces hansenii, Hansenula ciferrii, Hansenula polymorpha, Pichia polymorpha, Saccharomycopsis guttulata, and Saccharomyces chevalieri), isolated from various fruits have been examined for their organic growth factor requisites. H. ciferrii was completely deficient in thiamine, biotin, inositol, riboflavin, niacin, and partially deficient in pantothenic acid. It required an external supply of 0.1-1.0 ppm thiamine, 0.01-0.1 ppm biotin, 10.0 ppm inositol, 0.10 ppm niacin and riboflavin for its optimum growth. H. polymorpha showed partial deficiency only in xanthine. P. polymorpha gave indications of partial deficiencies in thiamine and biotin. S. guttulata was completely deficient in biotin, and partially deficient in adenine sulphate. It required 0.01 ppm biotin for optimum growth. S chevalieri was completely deficient in pyridoxine and partially deficient in thiamine. It required 0.1 ppm pyridoxine for maximum growth. D. hansenii and B bruxellensis were auxoautotrophic for the various growth factors studied. PMID:7242379

  15. Immune-Modulation by Epidermal Growth Factor Receptor Inhibitors: Implication on Anti-Tumor Immunity in Lung Cancer

    PubMed Central

    Herrmann, Amanda C.; Bernatchez, Chantale; Haymaker, Cara; Molldrem, Jeffrey J.; Hong, Waun Ki; Perez-Soler, Roman

    2016-01-01

    Skin toxicity is the most common toxicity caused by Epidermal Growth Factor Receptor (EGFR) inhibitors, and has been associated with clinical efficacy. As EGFR inhibitors enhance the expression of antigen presenting molecules in affected skin keratinocytes, they may concurrently facilitate neo-antigen presentation in lung cancer tumor cells contributing to anti-tumor immunity. Here, we investigated the modulatory effect of the EGFR inhibitor, erlotinib on antigen presenting molecules and PD-L1, prominent immune checkpoint protein, of skin keratinocytes and lung cancer cell lines to delineate the link between EGFR signaling pathway inhibition and potential anti-tumor immunity. Erlotinib up-regulated MHC-I and MHC-II proteins on IFNγ treated keratinocytes but abrogated IFNγ-induced expression of PD-L1, suggesting the potential role of infiltrating autoreactive T cells in the damage of keratinocytes in affected skin. Interestingly, the surface expression of MHC-I, MHC-II, and PD-L1 was up-regulated in response to IFNγ more often in lung cancer cell lines sensitive to erlotinib, but only expression of PD-L1 was inhibited by erlotinib. Further, erlotinib significantly increased T cell mediated cytotoxicity on lung cancer cells. Lastly, the analysis of gene expression dataset of 186 lung cancer cell lines from Cancer Cell Line Encyclopedia demonstrated that overexpression of PD-L1 was associated with sensitivity to erlotinib and higher expression of genes related to antigen presenting pathways and IFNγ signaling pathway. Our findings suggest that the EGFR inhibitors can facilitate anti-tumor adaptive immune responses by breaking tolerance especially in EGFR driven lung cancer that are associated with overexpression of PD-L1 and genes related to antigen presentation and inflammation. PMID:27467256

  16. Immune-Modulation by Epidermal Growth Factor Receptor Inhibitors: Implication on Anti-Tumor Immunity in Lung Cancer.

    PubMed

    Im, Jin S; Herrmann, Amanda C; Bernatchez, Chantale; Haymaker, Cara; Molldrem, Jeffrey J; Hong, Waun Ki; Perez-Soler, Roman

    2016-01-01

    Skin toxicity is the most common toxicity caused by Epidermal Growth Factor Receptor (EGFR) inhibitors, and has been associated with clinical efficacy. As EGFR inhibitors enhance the expression of antigen presenting molecules in affected skin keratinocytes, they may concurrently facilitate neo-antigen presentation in lung cancer tumor cells contributing to anti-tumor immunity. Here, we investigated the modulatory effect of the EGFR inhibitor, erlotinib on antigen presenting molecules and PD-L1, prominent immune checkpoint protein, of skin keratinocytes and lung cancer cell lines to delineate the link between EGFR signaling pathway inhibition and potential anti-tumor immunity. Erlotinib up-regulated MHC-I and MHC-II proteins on IFNγ treated keratinocytes but abrogated IFNγ-induced expression of PD-L1, suggesting the potential role of infiltrating autoreactive T cells in the damage of keratinocytes in affected skin. Interestingly, the surface expression of MHC-I, MHC-II, and PD-L1 was up-regulated in response to IFNγ more often in lung cancer cell lines sensitive to erlotinib, but only expression of PD-L1 was inhibited by erlotinib. Further, erlotinib significantly increased T cell mediated cytotoxicity on lung cancer cells. Lastly, the analysis of gene expression dataset of 186 lung cancer cell lines from Cancer Cell Line Encyclopedia demonstrated that overexpression of PD-L1 was associated with sensitivity to erlotinib and higher expression of genes related to antigen presenting pathways and IFNγ signaling pathway. Our findings suggest that the EGFR inhibitors can facilitate anti-tumor adaptive immune responses by breaking tolerance especially in EGFR driven lung cancer that are associated with overexpression of PD-L1 and genes related to antigen presentation and inflammation. PMID:27467256

  17. Activated protein C: A regulator of human skin epidermal keratinocyte function.

    PubMed

    McKelvey, Kelly; Jackson, Christopher John; Xue, Meilang

    2014-05-26

    Activated protein C (APC) is a physiological anticoagulant, derived from its precursor protein C (PC). Independent of its anticoagulation, APC possesses strong anti-inflammatory, anti-apoptotic and barrier protective properties which appear to be protective in a number of disorders including chronic wound healing. The epidermis is the outermost skin layer and provides the first line of defence against the external environment. Keratinocytes are the most predominant cells in the epidermis and play a critical role in maintaining epidermal barrier function. PC/APC and its receptor, endothelial protein C receptor (EPCR), once thought to be restricted to the endothelium, are abundantly expressed by skin epidermal keratinocytes. These cells respond to APC by upregulating proliferation, migration and matrix metalloproteinase-2 activity and inhibiting apoptosis/inflammation leading to a wound healing phenotype. APC also increases barrier function of keratinocyte monolayers by promoting the expression of tight junction proteins and re-distributing them to cell-cell contacts. These cytoprotective properties of APC are mediated through EPCR, protease-activated receptors, epidermal growth factor receptor or Tie2. Future preventive and therapeutic uses of APC in skin disorders associated with disruption of barrier function and inflammation look promising. This review will focus on APC's function in skin epidermis/keratinocytes and its therapeutical potential in skin inflammatory conditions.

  18. IL-22 inhibits epidermal differentiation and induces proinflammatory gene expression and migration of human keratinocytes.

    PubMed

    Boniface, Katia; Bernard, François-Xavier; Garcia, Martine; Gurney, Austin L; Lecron, Jean-Claude; Morel, Franck

    2005-03-15

    IL-22 belongs to a family of cytokines structurally related to IL-10, including IL-19, IL-20, IL-24, and IL-26. In contrast to IL-10, IL-22 has proinflammatory activities. IL-22 signals through a class II cytokine receptor composed of an IL-22-binding chain, IL-22RA1, and the IL-10RB subunit, which is shared with the IL-10R. In the present study, we show that short-term cultured human epidermal keratinocytes express a functional IL-22R but no IL-10R. Accordingly, IL-22 but not IL-10 induces STAT3 activation in keratinocytes. Using a cDNA array screening approach, real-time RT-PCR, and Western blot analysis, we demonstrate that IL-22 up-regulates, in a dose-dependent manner, the expression of S100A7, S100A8, S100A9, a group of proinflammatory molecules belonging to the S100 family of calcium-binding proteins, as well as the matrix metalloproteinase 3, the platelet-derived growth factor A, and the CXCL5 chemokine. In addition, IL-22 induces keratinocyte migration in an in vitro injury model and down-regulates the expression of at least seven genes associated with keratinocyte differentiation. Finally, we show that IL-22 strongly induces hyperplasia of reconstituted human epidermis. Taken together, these results suggest that IL-22 plays an important role in skin inflammatory processes and wound healing.

  19. High commitment of embryonic keratinocytes to terminal differentiation through a Notch1-caspase 3 regulatory mechanism.

    PubMed

    Okuyama, Ryuhei; Nguyen, Bach-Cuc; Talora, Claudio; Ogawa, Eisaku; Tommasi di Vignano, Alice; Lioumi, Maria; Chiorino, Giovanna; Tagami, Hachiro; Woo, Minna; Dotto, G Paolo

    2004-04-01

    Embryonic cells are expected to possess high growth/differentiation potential, required for organ morphogenesis and expansion during development. However, little is known about the intrinsic properties of embryonic epithelial cells due to difficulties in their isolation and cultivation. We report here that pure keratinocyte populations from E15.5 mouse embryos commit irreversibly to differentiation much earlier than newborn cells. Notch signaling, which promotes keratinocyte differentiation, is upregulated in embryonic keratinocyte and epidermis, and elevated caspase 3 expression, which we identify as a transcriptional Notch1 target, accounts in part for the high commitment of embryonic keratinocytes to terminal differentiation. In vivo, lack of caspase 3 results in increased proliferation and decreased differentiation of interfollicular embryonic keratinocytes, together with decreased activation of PKC-delta, a caspase 3 substrate which functions as a positive regulator of keratinocyte differentiation. Thus, a Notch1-caspase 3 regulatory mechanism underlies the intrinsically high commitment of embryonic keratinocytes to terminal differentiation.

  20. Factor-binding element in the human c-myc promoter involved in transcriptional regulation by transforming growth factor. beta. 1 and by the retinoblastoma gene product

    SciTech Connect

    Pietenpol, J.A.; Stein, R.W.; Moses, H.L. ); Muenger, K.; Howley, P.M. )

    1991-11-15

    Previous studies have shown that transforming growth factor {beta}1 (TGF-{beta}1) inhibition of keratinocyte proliferation involves suppression of c-myc transcription, and indirect evidence has suggested that the retinoblastoma gene product (pRB) may be involved in this process. In this study, transient expression of pRB in skin keratinocytes was shown to repress transcription of the human c-myc promoter region was required for regulation by both TGF-{beta}1 and pRB. These sequences, termed the TGF-{beta} control element (TCE), lie between positions {minus}86 and {minus}63 relative to the P1 transcription start site. Oligonucleotides containing the TCE bound to several nuclear factors in mobility-shift assays using extracts from cells with or without normal pRB. Binding of some factors was inhibited by TGF-{beta}1 treatment of TGF-{beta}-sensitive but not TGF-{beta}-insensitive cells. These data indicate that pRB can suppress c-myc transcription and growth inhibition.

  1. Placenta Growth Factor in Diabetic Wound Healing

    PubMed Central

    Cianfarani, Francesca; Zambruno, Giovanna; Brogelli, Laura; Sera, Francesco; Lacal, Pedro Miguel; Pesce, Maurizio; Capogrossi, Maurizio C.; Failla, Cristina Maria; Napolitano, Monica; Odorisio, Teresa

    2006-01-01

    Reduced microcirculation and diminished expression of growth factors contribute to wound healing impairment in diabetes. Placenta growth factor (PlGF), an angiogenic mediator promoting pathophysiological neovascularization, is expressed during cutaneous wound healing and improves wound closure by enhancing angiogenesis. By using streptozotocin-induced diabetic mice, we here demonstrate that PlGF induction is strongly reduced in diabetic wounds. Diabetic transgenic mice overexpressing PlGF in the skin displayed accelerated wound closure compared with diabetic wild-type littermates. Moreover, diabetic wound treatment with an adenovirus vector expressing the human PlGF gene (AdCMV.PlGF) significantly accelerated the healing process compared with wounds treated with a control vector. The analysis of treated wounds showed that PlGF gene transfer improved granulation tissue formation, maturation, and vascularization, as well as monocytes/macrophages local recruitment. Platelet-derived growth factor, fibroblast growth factor-2, and vascular endothelial growth factor mRNA levels were increased in AdCMV.PlGF-treated wounds, possibly enhancing PlGF-mediated effects. Finally, PlGF treatment stimulated cultured dermal fibroblast migration, pointing to a direct role of PlGF in accelerating granulation tissue maturation. In conclusion, our data indicate that reduced PlGF expression contributes to impaired wound healing in diabetes and that PlGF gene transfer to diabetic wounds exerts therapeutic activity by promoting different aspects of the repair process. PMID:17003476

  2. Distinct Effects of Different Phosphatidylglycerol Species on Mouse Keratinocyte Proliferation

    PubMed Central

    Xie, Ding; Seremwe, Mutsa; Edwards, John G.; Podolsky, Robert; Bollag, Wendy B.

    2014-01-01

    We have previously shown that liposomes composed of egg-derived phosphatidylglycerol (PG), with a mixed fatty acid composition (comprising mainly palmitate and oleate), inhibit the proliferation and promote the differentiation of rapidly dividing keratinocytes, and stimulate the growth of slowly proliferating epidermal cells. To determine the species of PG most effective at modulating keratinocyte proliferation, primary mouse keratinocytes were treated with different PG species, and proliferation was measured. PG species containing polyunsaturated fatty acids were effective at inhibiting rapidly proliferating keratinocytes, whereas PG species with monounsaturated fatty acids were effective at promoting proliferation in slowly dividing cells. Thus, palmitoyl-arachidonyl-PG (16∶0/20∶4), palmitoyl-linoleoyl-PG (16∶0/18∶2), dilinoleoyl-PG (18∶2/18∶2) and soy PG (a PG mixture with a large percentage of polyunsaturated fatty acids) were particularly effective at inhibiting proliferation in rapidly dividing keratinocytes. Conversely, palmitoyl-oleoyl-PG (16∶0/18∶1) and dioleoyl-PG (18∶1/18∶1) were especially effective proproliferative PG species. This result represents the first demonstration of opposite effects of different species of a single class of phospholipid and suggests that these different PG species may signal to diverse effector enzymes to differentially affect keratinocyte proliferation and normalize keratinocyte proliferation. Thus, different PG species may be useful for treating skin diseases characterized by excessive or insufficient proliferation. PMID:25233484

  3. Serum growth factors in asbestosis patients.

    PubMed

    Li, Yongliang; Karjalainen, Antti; Koskinen, Heikki; Vainio, Harri; Pukkala, Eero; Hemminki, Kari; Brandt-Rauf, Paul W

    2009-02-01

    Various growth factors, including platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-beta, have been implicated in the pathogenesis of asbestos-induced disease. PDGF and TGF-beta levels were determined by enzyme-linked immunosorbent assays in the banked serum samples of a cohort of workers with asbestosis, and the relationships of the growth factor levels to the subsequent development of cancer and to the radiographic severity and progression of asbestosis in the cohort were examined. Serum levels of PDGF and TGF-beta were found to be unrelated to the development of cancer, and serum levels of PDGF were found to be unrelated to the severity and progression of asbestosis. However, serum levels of TGF-beta were found to be statistically significantly related to disease severity (p = 0.01), increasing approximately 2.4-fold from ILO radiographic category 0 to category 3, and they were marginally related to disease progression (p = 0.07), in multivariate analysis controlling for other contributory factors including cumulative asbestos exposure. This suggests that serum TGF-beta may be a useful biomarker for asbestos-induced fibrotic disease. PMID:19283526

  4. Establishment of a 2-week canine skin organ culture model and its pharmacological modulation by epidermal growth factor and dexamethasone.

    PubMed

    Abramo, Francesca; Pirone, Andrea; Lenzi, Carla; Vannozzi, Iacopo; Della Valle, Maria Federica; Miragliotta, Vincenzo

    2016-09-01

    Although canine skin models are already available as either monocellular or organotypic cultures, they only partly recapitulate normal skin morphological features and function. The objective of this study was to establish a canine serum-free skin organ culture model and verify whether dexamethasone could rescue epidermal growth factor-induced changes. The study of morphological changes as a response to pharmacological substances may indeed help to investigate skin physiology and pathology. Normal skin was obtained from five client-owned dogs subjected to surgical procedures unrelated to dermatological conditions. Two experimental designs were performed: (i) two-week viability of the skin culture; (ii) dexamethasone (DMS) inhibition of epidermal growth factor (EGF)-induced effects. Serum-free submerged organ cultures were established in Williams' E medium supplemented with penicillin-streptomycin, insulin, hydrocortisone and l-glutamine. General morphological features of skin anatomical structures were well maintained up to day 14, scattered pyknotic nuclei were visible in the epidermis from day 7. Normal keratinocyte differentiation was confirmed by cytokeratin (K) 10, K14 and loricrin immunostaining. Epidermal thickness did not decrease throughout the study. A decrease in keratinocyte proliferation was observed at day 7 and 14. Treatment with EGF induced both keratinocyte proliferation and thickening of the epidermis; both responses were counteracted by DMS. Treatment with EGF increased the length of epithelial tongues at the edge of the skin explants; this effect was further enhanced by DMS supplementation. Our findings demonstrate the potential use of a full-thickness canine skin organ culture model for the study of skin physiology and pharmacological response to exogenous compounds, especially in the field of re-epithelialisation and keratinization disorders. PMID:27058637

  5. Epidermal Growth Factor and Intestinal Barrier Function.

    PubMed

    Tang, Xiaopeng; Liu, Hu; Yang, Shufen; Li, Zuohua; Zhong, Jinfeng; Fang, Rejun

    2016-01-01

    Epidermal growth factor (EGF) is a 53-amino acid peptide that plays an important role in regulating cell growth, survival, migration, apoptosis, proliferation, and differentiation. In addition, EGF has been established to be an effective intestinal regulator helping to protect intestinal barrier integrity, which was essential for the absorption of nutrients and health in humans and animals. Several researches have demonstrated that EGF via binding to the EGF receptor and subsequent activation of Ras/MAPK, PI3K/AKT, PLC-γ/PKC, and STATS signal pathways regulates intestinal barrier function. In this review, the relationship between epidermal growth factor and intestinal development and intestinal barrier is described, to provide a better understanding of the effects of EGF on intestine development and health. PMID:27524860

  6. Epidermal Growth Factor and Intestinal Barrier Function

    PubMed Central

    Liu, Hu; Yang, Shufen; Li, Zuohua; Zhong, Jinfeng

    2016-01-01

    Epidermal growth factor (EGF) is a 53-amino acid peptide that plays an important role in regulating cell growth, survival, migration, apoptosis, proliferation, and differentiation. In addition, EGF has been established to be an effective intestinal regulator helping to protect intestinal barrier integrity, which was essential for the absorption of nutrients and health in humans and animals. Several researches have demonstrated that EGF via binding to the EGF receptor and subsequent activation of Ras/MAPK, PI3K/AKT, PLC-γ/PKC, and STATS signal pathways regulates intestinal barrier function. In this review, the relationship between epidermal growth factor and intestinal development and intestinal barrier is described, to provide a better understanding of the effects of EGF on intestine development and health. PMID:27524860

  7. The epithelial-mesenchymal transition induced by keratinocyte growth conditions is overcome by E6 and E7 from HPV16, but not HPV8 and HPV38: Characterization of global transcription profiles

    SciTech Connect

    Azzimonti, Barbara; Dell'Oste, Valentina; Borgogna, Cinzia; Mondini, Michele; Gugliesi, Francesca; De Andrea, Marco; Chiorino, Giovanna; Scatolini, Maria; Ghimenti, Chiara; Landolfo, Santo; Gariglio, Marisa

    2009-06-05

    The aim of this study was to evaluate the growth properties of primary human keratinocytes expressing E6 and E7 proteins, which are from either the beta- or alpha-genotypes, under different culture conditions. We demonstrated that keratinocytes expressing E6 and E7, from both HPV8 and 38, irreversibly underwent the epithelial-mesenchymal transition (EMT) when grown on plastic with FAD medium (F12/DMEM/5%FBS). Expression of E6/E7 from HPV16 was capable of fully overcoming the FAD-induced EMT. Immortalization was only observed in HPV16-transduced cell lines, while the more proliferating phenotype of both KerHPV8 and 38 was mainly related to FAD-induced EMT. Microarray analysis of exponentially growing cells identified 146 cellular genes that were differentially regulated in HPV16 compared to HPV8- and 38-transduced cells. A large accumulation of transcripts associated with epidermal development and differentiation was observed in HPV16-transduced cells, whereas transcripts of genes involved in the extracellular matrix, multicellular organismal processes, and inflammatory response were affected in HPV8 and 38-transduced cells.

  8. Nerve growth factor promotes human hemopoietic colony growth and differentiation

    SciTech Connect

    Matsuda, H.; Coughlin, M.D.; Bienenstock, J.; Denburg, J.A. )

    1988-09-01

    Nerve growth factor (NGF) is a neurotropic polypeptide necessary for the survival and growth of some central neurons, as well as sensory afferent and sympathetic neurons. Much is now known of the structural and functional characteristics of NGF, whose gene has recently been clones. Since it is synthesized in largest amounts by the male mouse submandibular gland, its role exclusively in nerve growth is questionable. These experiments indicate that NGF causes a significant stimulation of granulocyte colonies grown from human peripheral blood in standard hemopoietic methylcellulose assays. Further, NGF appears to act in a relatively selective fashion to induce the differentiation of eosinophils and basophils/mast cells. Depletion experiments show that the NGF effect may be T-cell dependent and that NGF augments the colony-stimulating effect of supernatants from the leukemic T-cell (Mo) line. The hemopoietic activity of NGF is blocked by {sup 125}I-polyclonal and monoclonal antibodies to NGF. The authors conclude that NGF may indirectly act as a local growth factor in tissues other than those of the nervous system by causing T cells to synthesize or secrete molecules with colony-stimulating activity. In view of the synthesis of NGF in tissue injury, the involvement of basophils/mast cells and eosinophils in allergic and other inflammatory processes, and the association of mast cells with fibrosis and tissue repair, they postulate that NGF plays an important biological role in a variety of repair processes.

  9. Beta Adrenergic Receptors in Keratinocytes

    PubMed Central

    Sivamani, Raja K.; Lam, Susanne T.; Isseroff, R. Rivkah

    2007-01-01

    Synopsis Beta2 adrenergic receptors were identified in keratinocytes more than 30 years ago, but their function in the epidermis continues to be elucidated. Abnormalities in their expression, signaling pathway, or in the generation of endogenous catecholamine agonists by keratinocytes have been implicated in the pathogenesis of cutaneous diseases such as atopic dermatitis, vitiligo and psoriasis. New studies also indicate that the beta2AR also modulates keratinocyte migration, and thus can function to regulate wound re-epithelialization. This review focuses on the function of these receptors in keratinocytes and their contribution to cutaneous physiology and disease. PMID:17903623

  10. Vascular endothelial growth factor B, a novel growth factor for endothelial cells.

    PubMed Central

    Olofsson, B; Pajusola, K; Kaipainen, A; von Euler, G; Joukov, V; Saksela, O; Orpana, A; Pettersson, R F; Alitalo, K; Eriksson, U

    1996-01-01

    We have isolated and characterized a novel growth factor for endothelial cells, vascular endothelial growth factor B (VEGF-B), with structural similarities to vascular endothelial growth factor (VEGF) and placenta growth factor. VEGF-B was particularly abundant in heart and skeletal muscle and was coexpressed with VEGF in these and other tissues. VEGF-B formed cell-surface-associated disulfide-linked homodimers and heterodimerized with VEGF when coexpressed. Conditioned medium from transfected 293EBNA cells expressing VEGF-B stimulated DNA synthesis in endothelial cells. Our results suggest that VEGF-B has a role in angiogenesis and endothelial cell growth, particularly in muscle. Images Fig. 3 Fig. 4 Fig. 5 PMID:8637916

  11. Nerve Growth Factor and Diabetic Neuropathy

    PubMed Central

    Vinik, Aaron

    2003-01-01

    Neuropathy is one of the most debilitating complications of both type 1 and type 2 diabetes, with estimates of prevalence between 50–90% depending on the means of detection. Diabetic neuropathies are heterogeneous and there is variable involvement of large myelinated fibers and small, thinly myelinated fibers. Many of the neuronal abnormalities in diabetes can be duplicated by experimental depletion of specific neurotrophic factors, their receptors or their binding proteins. In experimental models of diabetes there is a reduction in the availability of these growth factors, which may be a consequence of metabolic abnormalities, or may be independent of glycemic control. These neurotrophic factors are required for the maintenance of the neurons, the ability to resist apoptosis and regenerative capacity. The best studied of the neurotrophic factors is nerve growth factor (NGF) and the related members of the neurotrophin family of peptides. There is increasing evidence that there is a deficiency of NGF in diabetes, as well as the dependent neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) that may also contribute to the clinical symptoms resulting from small fiber dysfunction. Similarly, NT3 appears to be important for large fiber and IGFs for autonomic neuropathy. Whether the observed growth factor deficiencies are due to decreased synthesis, or functional, e.g. an inability to bind to their receptor, and/or abnormalities in nerve transport and processing, remains to be established. Although early studies in humans on the role of neurotrophic factors as a therapy for diabetic neuropathy have been unsuccessful, newer agents and the possibilities uncovered by further studies should fuel clinical trials for several generations. It seems reasonable to anticipate that neurotrophic factor therapy, specifically targeted at different nerve fiber populations, might enter the therapeutic armamentarium. PMID:14668049

  12. Transcriptional profiling of ectoderm specification to keratinocyte fate in human embryonic stem cells.

    PubMed

    Tadeu, Ana Mafalda Baptista; Lin, Samantha; Hou, Lin; Chung, Lisa; Zhong, Mei; Zhao, Hongyu; Horsley, Valerie

    2015-01-01

    In recent years, several studies have shed light into the processes that regulate epidermal specification and homeostasis. We previously showed that a broad-spectrum γ-secretase inhibitor DAPT promoted early keratinocyte specification in human embryonic stem cells triggered to undergo ectoderm specification. Here, we show that DAPT accelerates human embryonic stem cell differentiation and induces expression of the ectoderm protein AP2. Furthermore, we utilize RNA sequencing to identify several candidate regulators of ectoderm specification including those involved in epithelial and epidermal development in human embryonic stem cells. Genes associated with transcriptional regulation and growth factor activity are significantly enriched upon DAPT treatment during specification of human embryonic stem cells to the ectoderm lineage. The human ectoderm cell signature identified in this study contains several genes expressed in ectodermal and epithelial tissues. Importantly, these genes are also associated with skin disorders and ectodermal defects, providing a platform for understanding the biology of human epidermal keratinocyte development under diseased and homeostatic conditions.

  13. [New aspects on the pathogenesis of cholesteatoma: the possible role of immune cell-induced keratinocyte hyperproliferation].

    PubMed

    Bujía, J; Holly, A; Kim, C; Schilling, V; Kastenbauer, E

    1993-06-01

    Cholesteatoma of the middle ear consists of keratinising squamous epithelium in the middle ear cavity. Many points of the pathogenesis of cholesteatoma seem to be related to immune cell infiltrates in stroma and to epithelial cell migration and proliferation. In our study we found that the vast majority of cells infiltrating the stroma consisted of T-cells and macrophages, showing an immunologically activated state. Furthermore, the cholesteatoma epithelium showed an enhanced simultaneous expression of keratine-16 and Ki-67 positive cells. This expression was accompanied by the over-expression of transforming growth factor (TGF)-alpha, and its receptor, epidermal growth factor receptor (EGF-R) and interleukin-1, suggesting that the proliferation of keratinocytes could be stimulated in an autocrine manner. Finally, we conclude that the presence of immunologically activated immune cells in the stroma may be responsible for keratinocyte dysregulation in cholesteatoma epithelium.

  14. UVB-irradiated keratinocytes induce melanoma-associated ganglioside GD3 synthase gene in melanocytes via secretion of tumor necrosis factor α and interleukin 6

    SciTech Connect

    Miyata, Maiko; Ichihara, Masatoshi; Tajima, Orie; Sobue, Sayaka; Kambe, Mariko; Sugiura, Kazumitsu; Furukawa, Koichi; Furukawa, Keiko

    2014-03-07

    Highlights: • Melanocytes showed low ST8SIA1 and high B3GALT4 levels in contrast with melanomas. • Direct UVB irradiation of melanocytes did not induce ganglioside synthase genes. • Culture supernatants of UVB-irradiated keratinocytes induced ST8SIA1 in melanocytes. • TNFα and IL-6 secreted from keratinocytes enhanced ST8SIA1 expression in melanocytes. • Inflammatory cytokines induced melanoma-related ST8SIA1 in melanocytes. - Abstract: Although expression of gangliosides and their synthetic enzyme genes in malignant melanomas has been well studied, that in normal melanocytes has been scarcely analyzed. In particular, changes in expression levels of glycosyltransferase genes responsible for ganglioside synthesis during evolution of melanomas from melanocytes are very important to understand roles of gangliosides in melanomas. Here, expression of glycosyltransferase genes related to the ganglioside synthesis was analyzed using RNAs from cultured melanocytes and melanoma cell lines. Quantitative RT-PCR revealed that melanomas expressed high levels of mRNA of GD3 synthase and GM2/GD2 synthase genes and low levels of GM1/GD1b synthase genes compared with melanocytes. As a representative exogenous stimulation, effects of ultraviolet B (UVB) on the expression levels of 3 major ganglioside synthase genes in melanocytes were analyzed. Although direct UVB irradiation of melanocytes caused no marked changes, culture supernatants of UVB-irradiated keratinocytes (HaCaT cells) induced definite up-regulation of GD3 synthase and GM2/GD2 synthase genes. Detailed examination of the supernatants revealed that inflammatory cytokines such as TNFα and IL-6 enhanced GD3 synthase gene expression. These results suggest that inflammatory cytokines secreted from UVB-irradiated keratinocytes induced melanoma-associated ganglioside synthase genes, proposing roles of skin microenvironment in the promotion of melanoma-like ganglioside profiles in melanocytes.

  15. Growth Factors and Tension-Induced Skeletal Muscle Growth

    NASA Technical Reports Server (NTRS)

    Vandenburgh, Herman H.

    1994-01-01

    The project investigated biochemical mechanisms to enhance skeletal muscle growth, and developed a computer based mechanical cell stimulator system. The biochemicals investigated in this study were insulin/(Insulin like Growth Factor) IGF-1 and Steroids. In order to analyze which growth factors are essential for stretch-induced muscle growth in vitro, we developed a defined, serum-free medium in which the differentiated, cultured avian muscle fibers could be maintained for extended periods of time. The defined medium (muscle maintenance medium, MM medium) maintains the nitrogen balance of the myofibers for 3 to 7 days, based on myofiber diameter measurements and myosin heavy chain content. Insulin and IGF-1, but not IGF-2, induced pronounced myofiber hypertrophy when added to this medium. In 5 to 7 days, muscle fiber diameters increase by 71 % to 98% compared to untreated controls. Mechanical stimulation of the avian muscle fibers in MM medium increased the sensitivity of the cells to insulin and IGF-1, based on a leftward shift of the insulin dose/response curve for protein synthesis rates. (54). We developed a ligand binding assay for IGF-1 binding proteins and found that the avian skeletal muscle cultures produced three major species of 31, 36 and 43 kD molecular weight (54) Stretch of the myofibers was found to have no significant effect on the efflux of IGF-1 binding proteins, but addition of exogenous collagen stimulated IGF-1 binding protein production 1.5 to 5 fold. Steroid hormones have a profound effect on muscle protein turnover rates in vivo, with the stress-related glucocorticoids inducing rapid skeletal muscle atrophy while androgenic steroids induce skeletal muscle growth. Exercise in humans and animals reduces the catabolic effects of glucocorticoids and may enhance the anabolic effects of androgenic steroids on skeletal muscle. In our continuing work on the involvement of exogenrus growth factors in stretch-induced avian skeletal muscle growth, we

  16. New detection methods of growth hormone and growth factors.

    PubMed

    Bidlingmaier, Martin

    2012-01-01

    Human growth hormone (GH), but also GH related growth factors like the insulin-like growth factor-1 (IGF-1) are known to be abused in sports. Although the scientific evidence supporting a distinct effect of GH on performance in healthy trained subjects is limited, it has been repeatedly found with athletes or trainers, and the recent introduction of a first test to detect GH doping has led to a number of positive cases. Currently, there is no test for the detection of IGF-1 introduced worldwide, but confiscation of the drug from sports teams can be taken as indirect evidence for its abuse. The major biochemical difficulty for the detection of GH is that the recombinant form is identical in physicochemical properties to the endogenous GH secreted by the pituitary gland. Furthermore, the very short half-life of GH in circulation inherently shortens the window of opportunity where the drug can be detected. Two strategies have been followed for more than a decade to develop a test to detect the application of recombinant GH: the marker approach, which is based on the elevation of GH-dependent markers above the level seen under physiological conditions evoked by administration of recombinant GH, and the isoform approach, which is based on a change in the pattern of GH isoforms in circulation following the injection of recombinant GH.

  17. Dual delivery of vascular endothelial growth factor and hepatocyte growth factor coacervate displays strong angiogenic effects.

    PubMed

    Awada, Hassan K; Johnson, Noah R; Wang, Yadong

    2014-05-01

    Controlled delivery of multiple growth factors (GFs) holds great potential for the clinical treatment of ischemic diseases and might be more therapeutically effective to reestablish vasculature than the provision of a single GF. Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) are two potent angiogenic factors. However, due to rapid degradation and dilution in the body, their clinical potential will rely on an effective mode of delivery. A coacervate, composed of heparin and a biodegradable polycation, which protects GFs from proteolysis and potentiates their bioactivities, is developed. Here, the coacervate incorporates VEGF and HGF and sustains their release for at least three weeks. Their strong angiogenic effects on endothelial cell proliferation and tube formation in vitro are confirmed. Furthermore, it is demonstrated that coacervate-based delivery of these factors has stronger effects than free application of both factors and to coacervate delivery of each GF separately.

  18. Growth hormone, insulin-like growth factor system and carcinogenesis.

    PubMed

    Boguszewski, Cesar Luiz; Boguszewski, Margaret Cristina da Silva; Kopchick, John J

    2016-01-01

    The growth hormone (GH) and insulin-like growth factor (IGF) system plays an important role in the regulation of cell proliferation, differentiation, apoptosis, and angiogenesis. In terms of cell cycle regulation, the GH-IGF system induces signalling pathways for cell growth that compete with other signalling systems that result in cell death; thus the final effect of these opposed forces is critical for normal and abnormal cell growth. The association of the GH-IGF system with carcinogenesis has long been hypothesised, mainly based on in vitro studies and the use of a variety of animal models of human cancer, and also on epidemiological and clinical evidence in humans. While ample experimental evidence supports a role of the GH-IGF system in tumour promotion and progression, with several of its components being currently tested as central targets for cancer therapy, the strength of evidence from patients with acromegaly, GH deficiency, or treated with GH is much weaker. In this review, we will attempt to consolidate this data. (Endokrynol Pol 2016; 67 (4): 414-426). PMID:27387246

  19. Migration of keratinocytes through tunnels of digested fibrin

    NASA Astrophysics Data System (ADS)

    Ronfard, Vincent; Barrandon, Yann

    2001-04-01

    We report here a hitherto undescribed form of cell migration. When a suspension of human keratinocytes is plated on a fibrin matrix, single cells invade the matrix and progress through it as rounded cells by dissolving the fibrin and thereby creating tunnels. These tunnels are cylindrical or helical, the latter being the result of constant change in the path of cellular advance around the helical axis. Helical tunnel formation is strongly promoted by epidermal growth factor. The rate of migration of the cell through the track of a helical tunnel (up to 2.1 mm per day) is about 7-fold greater than through a cylindrical tunnel. Pericellular fibrinolysis leading to tunnel formation depends on the presence of plasminogen in the medium and its conversion to plasmin by a cellular activator. Formation of tunnels requires that plasminogen activator be localized on the advancing surface of the keratinocyte; we propose that the tunnel is cylindrical when the site of release of plasmin is located at a fixed point on the cell surface and helical when the site of release precesses.

  20. Altered (/sup 125/I)epidermal growth factor binding and receptor distribution in psoriasis

    SciTech Connect

    Nanney, L.B.; Stoscheck, C.M.; Magid, M.; King, L.E. Jr.

    1986-03-01

    Stimulation of growth and differentiation of human epidermis by epidermal growth factor (EGF) is mediated by its binding to specific receptors. Whether EGF receptors primarily mediate cell division or differentiation in hyperproliferative disease such as psoriasis vulgaris is unclear. To study the pathogenesis of psoriasis, 4-mm2 punch biopsy specimens of normal, uninvolved, and involved psoriatic skin were assayed for EGF receptors by autoradiographic, immunohistochemical, and biochemical methods. Using autoradiographic and immunohistochemical methods, basal keratinocytes were found to contain the greatest number of EGF binding sites and immunoreactive receptors as compared to the upper layers of the epidermis in both normal epidermis and psoriatic skin. No EGF receptor differences between normal and psoriatic epidermis were observed in this layer. In the upper layers of the epidermis, a 2-fold increase in EGF binding capacity was observed in psoriatic skin as compared with normal thin or thick skin. Biochemical methods indicated that (/sup 125/I)EGF binding was increased in psoriatic epidermis as compared with similar thickness normal epidermis when measured on a protein basis. Epidermal growth factor was shown to increase phosphorylation of the EGF receptor in skin. EGF receptors retained in the nonmitotic stratum spinosum and parakeratotic stratum corneum may reflect the incomplete, abnormal differentiation that occurs in active psoriatic lesions. Alternatively, retained EGF receptors may play a direct role in inhibiting cellular differentiation in the suprabasal layers.

  1. Epidermal growth factor receptor signaling in tissue

    SciTech Connect

    Shvartsman, Stanislav; Wiley, H. S.; Lauffenburger, Douglas A.

    2004-08-01

    Abstract: A peptide purified from the salivary gland of a mouse was shown few years ago to accelerate incisor eruption and eyelid opening in newborn mice, and was named epidermal growth factor (EGF). The members of this family of peptide growth factors had been identified in numerous physiological and pathological contexts. EGF binds to a cell surface EGF receptor, which induces a biochemical modification (phosphorylation) of the receptor's cytoplasmic tail. There is a growing consensus in the research community that, in addition to cellular and molecular studies, the dynamics of the EGFR network and its operation must be examined in tissues. A key challenge is to integrate the existing molecular and cellular information into a system-level description of the EGFR network at the tissue and organism level. In this paper, the two examples of EGFR signaling in tissues are described, and the recent efforts to model EGFR autocrine loops, which is a predominant mode of EGFR activation in vivo, are summarized.

  2. Transforming growth factor-beta requires its target plasminogen activator inhibitor-1 for cytostatic activity.

    PubMed

    Kortlever, Roderik M; Nijwening, Jeroen H; Bernards, René

    2008-09-01

    The cytokine transforming growth factor beta (TGFbeta) has strong antiproliferative activity in most normal cells but contributes to tumor progression in the later stages of oncogenesis. It is not fully understood which TGFbeta target genes are causally involved in mediating its cytostatic activity. We report here that suppression of the TGFbeta target gene encoding plasminogen activator inhibitor-1 (PAI-1) by RNA interference leads to escape from the cytostatic activity of TGFbeta both in human keratinocytes (HaCaTs) and primary mouse embryo fibroblasts. Consistent with this, PAI-1 knock-out mouse embryo fibroblasts are also resistant to TGFbeta growth arrest. Conversely, we show that ectopic expression of PAI-1 in proliferating HaCaT cells induces a growth arrest. PAI-1 knockdown does not interfere with canonical TGFbeta signaling as judged by SMAD phosphorylation and induction of bona fide TGFbeta target genes. Instead, knockdown of PAI-1 results in sustained activation of protein kinase B. Significantly, we find that constitutive protein kinase B activity leads to evasion of the growth-inhibitory action of TGFbeta. Our data are consistent with a model in which induction of PAI-1 by TGFbeta is critical for the induction of proliferation arrest.

  3. Death penalty for keratinocytes: apoptosis versus cornification.

    PubMed

    Lippens, S; Denecker, G; Ovaere, P; Vandenabeele, P; Declercq, W

    2005-11-01

    Homeostasis implies a balance between cell growth and cell death. This balance is essential for the development and maintenance of multicellular organisms. Homeostasis is controlled by several mechanisms including apoptosis, a process by which cells condemned to death are completely eliminated. However, in some cases, total destruction and removal of dead cells is not desirable, as when they fulfil a specific function such as formation of the skin barrier provided by corneocytes, also known as terminally differentiated keratinocytes. In this case, programmed cell death results in accumulation of functional cell corpses. Previously, this process has been associated with apoptotic cell death. In this overview, we discuss differences and similarities in the molecular regulation of epidermal programmed cell death and apoptosis. We conclude that despite earlier confusion, apoptosis and cornification occur through distinct molecular pathways, and that possibly antiapoptotic mechanisms are implicated in the terminal differentiation of keratinocytes.

  4. Milk Epidermal Growth Factor and Gut Protection

    PubMed Central

    Dvorak, Bohuslav

    2010-01-01

    Maternal milk is a complex fluid with multifunctional roles within the developing gastrointestinal tract. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) are members of the family of EGF-related peptides. Biological actions of these growth factors are mediated via interaction with the EGF-receptor (EGF-R). In the early postnatal period, breast milk is the major source of EGF for the developing intestinal mucosa. HB-EGF is also detected in breast milk, but in concentrations 2 to 3 times lower than EGF. Under normal physiological conditions, the intestinal epithelium undergoes a continuing process of cell proliferation, differentiation and maturation. EGF plays an important role in these processes. In pathophysiologic situations, EGF contributes to epithelial protection from injury and post-injury mucosal repair. Necrotizing enterocolitis (NEC) is a devastating disease affecting prematurely born infants. The pathogenesis of NEC is not known and there is no effective treatment for this disease. In an experimental NEC model, oral administration of a physiological dose of EGF significantly reduces the incidence and severity of NEC. HB-EGF provides similar protection against NEC, but only when pharmacological doses are used. Further studies are necessary before EGF can be introduced as an efficient therapeutic approach of intestinal injury. PMID:20105663

  5. The linear C-terminal regions of epidermal growth factor (EGF) and transforming growth factor-alpha bind to different epitopes on the human EGF receptor.

    PubMed Central

    Lenferink, A E; De Roos, A D; Van Vugt, M J; Van de Poll, M L; Van Zoelen, E J

    1998-01-01

    Epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) bind with similar affinities in a competitive fashion to the human EGF receptor, and basically induce similar mitogenic responses. In spite of the fact that EGF and TGFalpha are structurally alike, it is still not clear if the two growth factors bind the receptor in an identical manner. The observation that the 13A9 antibody blocks binding of TGFalpha, but not that of EGF, to the human EGF receptor [Winkler, O'Connor, Winget and Fendly (1989) Biochemistry 28, 6373-6378] suggests that their binding characteristics are not identical. In the present study we have made use of a set of EGF/TGFalpha chimaeric molecules to show that the 13A9 antibody blocks receptor binding of ligands with TGFalpha sequences, but not of ligands with EGF sequences, in their C-terminal linear regions. Using HaCaT human keratinocyte cells in culture, it was determined that ligands that are able to bind the EGF receptor in the presence of 13A9 are also able to induce calcium release from intracellular stores in these cells, indicating that these ligands have the ability to activate the EGF receptor in the presence of the antibody. From these data it is concluded that the flexible C-terminal linear domains of EGF and TGFalpha bind to separate sequences on the EGF receptor, such that the binding domain of TGFalpha, but not that of EGF, overlaps with the binding epitope of the 13A9 antibody. PMID:9806896

  6. Post-transcriptional Regulation of Keratinocyte Progenitor Cell Expansion, Differentiation and Hair Follicle Regression by miR-22.

    PubMed

    Yuan, Shukai; Li, Feifei; Meng, Qingyong; Zhao, Yiqiang; Chen, Lei; Zhang, Hongquan; Xue, Lixiang; Zhang, Xiuqing; Lengner, Christopher; Yu, Zhengquan

    2015-05-01

    Hair follicles (HF) undergo precisely regulated recurrent cycles of growth, cessation, and rest. The transitions from anagen (growth), to catagen (regression), to telogen (rest) involve a physiological involution of the HF. This process is likely coordinated by a variety of mechanisms including apoptosis and loss of growth factor signaling. However, the precise molecular mechanisms underlying follicle involution after hair keratinocyte differentiation and hair shaft assembly remain poorly understood. Here we demonstrate that a highly conserved microRNA, miR-22 is markedly upregulated during catagen and peaks in telogen. Using gain- and loss-of-function approaches in vivo, we find that miR-22 overexpression leads to hair loss by promoting anagen-to-catagen transition of the HF, and that deletion of miR-22 delays entry to catagen and accelerates the transition from telogen to anagen. Ectopic activation of miR-22 results in hair loss due to the repression a hair keratinocyte differentiation program and keratinocyte progenitor expansion, as well as promotion of apoptosis. At the molecular level, we demonstrate that miR-22 directly represses numerous transcription factors upstream of phenotypic keratin genes, including Dlx3, Foxn1, and Hoxc13. We conclude that miR-22 is a critical post-transcriptional regulator of the hair cycle and may represent a novel target for therapeutic modulation of hair growth.

  7. The suppression of fibroblast growth factor 2/fibroblast growth factor 4-dependent tumour angiogenesis and growth by the anti-growth factor activity of dextran derivative (CMDB7).

    PubMed Central

    Bagheri-Yarmand, R.; Kourbali, Y.; Mabilat, C.; Morère, J. F.; Martin, A.; Lu, H.; Soria, C.; Jozefonvicz, J.; Crépin, M.

    1998-01-01

    Our previous studies showed that carboxymethyl benzylamide dextran (CMDB7) blocks basic fibroblast growth factor (FGF-2)-dependent cell proliferation of a human breast epithelial line (HBL100), suggesting its potential role as a potent antiangiogenic substance. The derived cell line (HH9), which was transformed with the hst/FGF4 gene, has been shown to be highly proliferative in vitro and to induce angiogenic tumours in nude mice. We show here that CMDB7 inhibits the mitogenic activities of the conditioned media from HBL 100 and HH9 cells in a dose-dependent manner. When HH9 cells were injected s.c. into nude mice, CMDB7 treatment (300 mg kg(-1) week(-1)) suppressed the tumour take and the tumour growth by about 50% and 80% respectively. Immunohistochemical analysis showed a highly significant decrease, by more than threefold, in the endothelial density of viable tumour regions, together with a significant increase in the necrosis area. This antiangiogenic activity of CMDB7 was further demonstrated by direct inhibition of calf pulmonary artery (CPAE) and human umbilical vein (HUVEC) endothelial cell proliferation and migration in vitro. In addition, we showed that CMDB7 inhibits specifically the mitogenic effects of the growth factors that bind to heparin such as FGF-2, FGF-4, platelet-derived growth factor (PDGF-BB) and transforming growth factor (TGF-beta1), but not those of epidermal growth factor (EGF) and insulin-like growth factor (IGF-1). These results demonstrate that CMDB7 inhibits FGF-2/FGF-4-dependent tumour growth and angiogenesis, most likely by disrupting the autocrine and paracrine effects of growth factors released from the tumour cells. Images Figure 4 PMID:9662260

  8. Growth Hormone and Insulin-Like Growth Factor-1.

    PubMed

    Nicholls, Adam R; Holt, Richard I G

    2016-01-01

    Human growth hormone (GH) was first isolated from the human pituitary gland in 1945 and found to promote the growth of children with hypopituitarism. Since the formation of the World Anti-Doping Association, human GH has appeared on the list of forbidden substances. There is a significant amount of anecdotal evidence that human GH is misused by athletes to enhance performance, and there have been a number of high-profile cases of GH use in professional sport. GH secretagogues (GH-Ss), which increase GH secretion, and insulin-like growth factor (IGF-1), which mediates many of the effects of GH, are also misused, although there is less evidence for this. The effectiveness of GH, IGF-1, and GH-Ss as performance-enhancing drugs remains unclear. Evidence from studies of GH use in people with hypopituitarism show several desirable outcomes, including increased lean body mass, increased strength, and increased exercise capacity. These anabolic and metabolic properties, coupled with the difficulty in detecting them, make them attractive as agents of misuse. Studies in healthy young adults have also demonstrated a performance benefit with GH and IGF-1. PMID:27347885

  9. Upregulation of miR-203 and miR-210 affect growth and differentiation of keratinocytes after exposure to sulfur mustard in normoxia and hypoxia.

    PubMed

    Deppe, Janina; Steinritz, Dirk; Santovito, Donato; Egea, Virginia; Schmidt, Annette; Weber, Christian; Ries, Christian

    2016-02-26

    Exposure of the skin to sulfur mustard (SM) results in long-term complications such as impaired tissue regeneration. Previous own studies in normal human epidermal keratinocytes (NHEK) treated with SM demonstrated reduced proliferation, premature differentiation and a restricted functionality of hypoxia-mediated signaling in the cells. Here, we investigated the involvement of microRNAs, miR-203 and miR-210, in these mechanisms. SM significantly upregulated the expression of miR-203 in NHEK when cultivated under normoxic and hypoxic conditions. SM had no effect on miR-210 under normoxia. However, miR-210 levels were greatly increased in NHEK when grown in hypoxia and further elevated upon exposure of the cells to SM. In normoxia and hypoxia, inhibition of miR-203 by transfection of NHEK with complementary oligonucleotides, anti-miR-203, attenuated the SM-induced impairment of metabolic activity and proliferation, and counteracted SM-promoted keratin-1 expression in these cells. Consistent ameliorating effects on dysregulated metabolic activity, proliferation and keratin-1 expression in SM-treated NHEK were obtained upon inhibition of miR-210 in these cells grown in hypoxia. Our findings provide evidence that miR-203 and miR-210 are key regulators in normal and SM-impaired keratinocyte functionality, and suggest potential usefulness of inhibitors against miR-203 and miR-210 for target-directed therapeutical intervention to improve re-epithelialization of SM-injured skin.

  10. P-cadherin regulates human hair growth and cycling via canonical Wnt signaling and transforming growth factor-β2.

    PubMed

    Samuelov, Liat; Sprecher, Eli; Tsuruta, Daisuke; Bíró, Tamás; Kloepper, Jennifer E; Paus, Ralf

    2012-10-01

    P-cadherin is a key component of epithelial adherens junctions, and it is prominently expressed in the hair follicle (HF) matrix. Loss-of-function mutations in CDH3, which encodes P-cadherin, result in hypotrichosis with juvenile macular dystrophy (HJMD), an autosomal recessive disorder featuring sparse and short hair. Here, we attempted to recapitulate some aspects of HJMD in vitro by transfecting normal, organ-cultured human scalp HFs with lipofectamine and CDH3-specific or scrambled control siRNAs. As in HJMD patients, P-cadherin silencing inhibited hair shaft growth, prematurely induced HF regression (catagen), and inhibited hair matrix keratinocyte proliferation. In situ, membrane β-catenin expression and transcription of the β-catenin target gene, axin2, were significantly reduced, whereas glycogen synthase kinase 3 β (GSK3β) and phospho-β-catenin immunoreactivity were increased. These effects were partially reversed by inhibiting GSK3β. P-cadherin silencing reduced the expression of the anagen-promoting growth factor, IGF-1, whereas that of transforming growth factor β 2 (TGFβ2; catagen promoter) was enhanced. Neutralizing TGFβ antagonized the catagen-promoting effects of P-cadherin silencing. In summary, we introduce human HFs as an attractive preclinical model for studying the functions of P-cadherin in human epithelial biology and pathology. This model demonstrates that cadherins can be successfully knocked down in an intact human organ in vitro, and shows that P-cadherin is needed for anagen maintenance by regulating canonical Wnt signaling and suppressing TGFβ2.

  11. Recombinant human epidermal growth factor precursor is a glycosylated membrane protein with biological activity.

    PubMed Central

    Mroczkowski, B; Reich, M; Chen, K; Bell, G I; Cohen, S

    1989-01-01

    NIH 3T3 cells were transfected with cDNA corresponding to human kidney prepro-epidermal growth factor (preproEGF) under control of the inducible mouse metallothionein promoter. The synthesis of recombinant human EGF precursor by these cells has provided us with a model system for analysis of the structure and activity of this precursor. In transfected cells, the precursor was present as an intrinsic 170-kilodalton membrane protein as well as a soluble protein in the extracellular medium; both forms were N glycosylated. Glycosylation of the EGF precursor was determined by (i) the direct incorporation of [3H]mannose and [3H]glucosamine, (ii) metabolic labeling in the presence or absence of glycosylation inhibitors, (iii) enzymatic cleavage of the precursor by N-glycanase or endoglycosidase II, and (iv) lectin chromatography. Recombinant human preproEGF was purified by affinity chromatography, using wheat germ lectin and antibodies to human EGF. The intact precursor was biologically active. Purified preparations of preproEGF (i) competed with 125I-labeled EGF for binding to the EGF receptor in intact fibroblast cells, (ii) activated the intrinsic tyrosine kinase activity of the EGF receptor in membrane preparations, and (iii) sustained the growth of a mouse keratinocyte cell line that is dependent on EGF for growth. These results suggest that proteolytic processing of the precursor may not be essential for its biological function. Images PMID:2789334

  12. Anti-epidermal growth factor receptor skin toxicity: a matter of topical hydration.

    PubMed

    Ferrari, Daris; Codecà, Carla; Bocci, Barbara; Crepaldi, Francesca; Violati, Martina; Viale, Giulia; Careri, Carmela; Caldiera, Sarah; Bordin, Veronica; Luciani, Andrea; Zonato, Sabrina; Cassinelli, Gabriela; Foa, Paolo

    2016-02-01

    Skin toxicity is a frequent complication of anti-epidermal growth factor receptor therapy, which can be an obstacle in maintaining the dose intensity and may negatively impact on the clinical outcome of cancer patients. Skin lesions depend on the disruption of the keratinocyte development pathways and no treatment is clearly effective in resolving the cutaneous alterations frequently found during anti-epidermal growth factor receptor therapy. Among systemic treatments, oral tetracycline proved to be useful in preventing skin manifestations. We describe the case of a patient affected by metastatic colorectal cancer, for whom a combination of chemotherapy and cetuximab was used as second-line treatment. The patient developed a symptomatic papulopustular skin rash that disappeared completely after a twice-daily application of a hydrating and moisturizing cream, mainly consisting of a mixture of paraffin, silicone compounds, and macrogol. The marked cutaneous amelioration allowed the patient to continue cetuximab without any further symptoms and was associated with a partial radiological response. PMID:26469836

  13. Human papilloma virus DNAs immortalize normal human mammary epithelial cells and reduce their growth factor requirements

    SciTech Connect

    Band, V.; Zajchowski, D.; Kulesa, V.; Sager, R. )

    1990-01-01

    Human papilloma virus (HPV) types 16 and 18 are most commonly associated with cervical carcinoma in patients and induce immortalization of human keratinocytes in culture. HPV has not been associated with breast cancer. This report describes the immortalization of normal human mammary epithelial cells (76N) by plasmid pHPV18 or pHPV16, each containing the linearized viral genome. Transfectants were grown continuously for more than 60 passages, whereas 76N cells senesce after 18-20 passages. The transfectants also differ from 76N cells in cloning in a completely defined medium called D2 and growing a minimally supplemented defined medium (D3) containing epidermal growth factor. All transfectant tested contain integrated HPV DNA, express HPV RNA, and produce HPV E7 protein. HPV transfectants do not form tumors in a nude mouse assay. It is concluded that products of the HPV genome induce immortalization of human breast epithelial cells and reduce their growth factor requirements. This result raises the possibility that HPV might be involved in breast cancer. Furthermore, other tissue-specific primary epithelial cells that are presently difficult to grown and investigate may also be immortalized by HPV.

  14. Nicotinamide downregulates gene expression of interleukin-6, interleukin-10, monocyte chemoattractant protein-1, and tumour necrosis factor-α gene expression in HaCaT keratinocytes after ultraviolet B irradiation.

    PubMed

    Monfrecola, G; Gaudiello, F; Cirillo, T; Fabbrocini, G; Balato, A; Lembo, S

    2013-03-01

    Ultraviolet (UV) radiation has profound effects on human skin, causing sunburn, inflammation, cellular-tissue injury, cell death, and skin cancer. Most of these effects are mediated by a number of cytokines produced by keratinocytes. In this study we investigated whether nicotinamide (NCT), the amide form of vitamin B3, might have a protective function in reducing the expression of interleukin (IL)-1β, IL-6, IL-8, IL-10, monocyte chemoattractant protein (MCP)-1 and tumour necrosis factor (TNF)-α in UV-irradiated keratinocytes. HaCaT cells were treated with UVB in the presence or absence of NCT, and cytokine mRNA levels were examined by quantitative real-time PCR. NCT significantly downregulated IL-6, IL-10, MCP-1 and TNF-α mRNA expression, whereas it did not exert any significant effect on IL-1β or IL-8 expression. Because of its ability to decrease these cytokine mediators after UV exposure, NCT is a possible therapy to improve or prevent conditions induced or aggravated by UV light.

  15. Decorin: A Growth Factor Antagonist for Tumor Growth Inhibition

    PubMed Central

    Järvinen, Tero A. H.; Prince, Stuart

    2015-01-01

    Decorin (DCN) is the best characterized member of the extracellular small leucine-rich proteoglycan family present in connective tissues, typically in association with or “decorating” collagen fibrils. It has substantial interest to clinical medicine owing to its antifibrotic, anti-inflammatory, and anticancer effects. Studies on DCN knockout mice have established that a lack of DCN is permissive for tumor development and it is regarded as a tumor suppressor gene. A reduced expression or a total disappearance of DCN has been reported to take place in various forms of human cancers during tumor progression. Furthermore, when used as a therapeutic molecule, DCN has been shown to inhibit tumor progression and metastases in experimental cancer models. DCN affects the biology of various types of cancer by targeting a number of crucial signaling molecules involved in cell growth, survival, metastasis, and angiogenesis. The active sites for the neutralization of different growth factors all reside in different parts of the DCN molecule. An emerging concept that multiple proteases, especially those produced by inflammatory cells, are capable of cleaving DCN suggests that native DCN could be inactivated in a number of pathological inflammatory conditions. In this paper, we review the role of DCN in cancer. PMID:26697491

  16. UVB-irradiated keratinocytes induce melanoma-associated ganglioside GD3 synthase gene in melanocytes via secretion of tumor necrosis factor α and interleukin 6.

    PubMed

    Miyata, Maiko; Ichihara, Masatoshi; Tajima, Orie; Sobue, Sayaka; Kambe, Mariko; Sugiura, Kazumitsu; Furukawa, Koichi; Furukawa, Keiko

    2014-03-01

    Although expression of gangliosides and their synthetic enzyme genes in malignant melanomas has been well studied, that in normal melanocytes has been scarcely analyzed. In particular, changes in expression levels of glycosyltransferase genes responsible for ganglioside synthesis during evolution of melanomas from melanocytes are very important to understand roles of gangliosides in melanomas. Here, expression of glycosyltransferase genes related to the ganglioside synthesis was analyzed using RNAs from cultured melanocytes and melanoma cell lines. Quantitative RT-PCR revealed that melanomas expressed high levels of mRNA of GD3 synthase and GM2/GD2 synthase genes and low levels of GM1/GD1b synthase genes compared with melanocytes. As a representative exogenous stimulation, effects of ultraviolet B (UVB) on the expression levels of 3 major ganglioside synthase genes in melanocytes were analyzed. Although direct UVB irradiation of melanocytes caused no marked changes, culture supernatants of UVB-irradiated keratinocytes (HaCaT cells) induced definite up-regulation of GD3 synthase and GM2/GD2 synthase genes. Detailed examination of the supernatants revealed that inflammatory cytokines such as TNFα and IL-6 enhanced GD3 synthase gene expression. These results suggest that inflammatory cytokines secreted from UVB-irradiated keratinocytes induced melanoma-associated ganglioside synthase genes, proposing roles of skin microenvironment in the promotion of melanoma-like ganglioside profiles in melanocytes. PMID:24548412

  17. UVB-irradiated keratinocytes induce melanoma-associated ganglioside GD3 synthase gene in melanocytes via secretion of tumor necrosis factor α and interleukin 6.

    PubMed

    Miyata, Maiko; Ichihara, Masatoshi; Tajima, Orie; Sobue, Sayaka; Kambe, Mariko; Sugiura, Kazumitsu; Furukawa, Koichi; Furukawa, Keiko

    2014-03-01

    Although expression of gangliosides and their synthetic enzyme genes in malignant melanomas has been well studied, that in normal melanocytes has been scarcely analyzed. In particular, changes in expression levels of glycosyltransferase genes responsible for ganglioside synthesis during evolution of melanomas from melanocytes are very important to understand roles of gangliosides in melanomas. Here, expression of glycosyltransferase genes related to the ganglioside synthesis was analyzed using RNAs from cultured melanocytes and melanoma cell lines. Quantitative RT-PCR revealed that melanomas expressed high levels of mRNA of GD3 synthase and GM2/GD2 synthase genes and low levels of GM1/GD1b synthase genes compared with melanocytes. As a representative exogenous stimulation, effects of ultraviolet B (UVB) on the expression levels of 3 major ganglioside synthase genes in melanocytes were analyzed. Although direct UVB irradiation of melanocytes caused no marked changes, culture supernatants of UVB-irradiated keratinocytes (HaCaT cells) induced definite up-regulation of GD3 synthase and GM2/GD2 synthase genes. Detailed examination of the supernatants revealed that inflammatory cytokines such as TNFα and IL-6 enhanced GD3 synthase gene expression. These results suggest that inflammatory cytokines secreted from UVB-irradiated keratinocytes induced melanoma-associated ganglioside synthase genes, proposing roles of skin microenvironment in the promotion of melanoma-like ganglioside profiles in melanocytes.

  18. mTOR inhibition by rapamycin increases ceramide synthesis by promoting transforming growth factor-β1/Smad signaling in the skin.

    PubMed

    Yamane, Takumi; Muramatsu, Aimi; Yoshino, Sawako; Matsui, Sho; Shimura, Mari; Tsujii, Yoshimasa; Iwatsuki, Ken; Kobayashi-Hattori, Kazuo; Oishi, Yuichi

    2016-04-01

    Although mammalian target of rapamycin (mTOR) mediates a wide variety of biological functions, little information is available on the effect of mTOR on the functions of skin cells. In this study, we investigated effects of mTOR inhibition by rapamycin on ceramide synthesis in the skin of rats and human keratinocytes and its regulatory mechanisms. The phosphorylation of p70 S6 kinase, which indicates mTOR activation, was induced in the skin of rats fed a high-fat diet, but this abnormality was reversed by supplementation with rapamycin. Ceramide levels and the mRNA levels of serine palmitoyltransferase (SPT) and transforming growth factor (TGF)-β1 were suppressed in the skin of rats fed high-fat diets, but this abnormality was reversed by supplementation with rapamycin. TGF-β1-induced SPT mRNA expression was blocked by SB525334, an inhibitor of TGF-β1-induced Smad2/3 nuclear localization, in human keratinocytes. Rapamycin-induced SPT mRNA expression was blocked by an anti-TGF-β1 antibody or SB525334 in human keratinocytes. These results show that mTOR inhibition by rapamycin increases ceramide synthesis by promoting TGF-β1/Smad signaling in the skin. PMID:27239444

  19. Transcription-dependent epidermal growth factor receptor activation by hepatocyte growth factor.

    PubMed

    Reznik, Thomas E; Sang, Yingying; Ma, Yongxian; Abounader, Roger; Rosen, Eliot M; Xia, Shuli; Laterra, John

    2008-01-01

    The mechanisms and biological implications of coordinated receptor tyrosine kinase coactivation remain poorly appreciated. Epidermal growth factor receptor (EGFR) and c-Met are frequently coexpressed in cancers, including those associated with hepatocyte growth factor (HGF) overexpression, such as malignant astrocytoma. In a previous analysis of the HGF-induced transcriptome, we found that two EGFR agonists, transforming growth factor-alpha and heparin-binding epidermal growth factor-like growth factor (HB-EGF), are prominently up-regulated by HGF in human glioma cells. We now report that stimulating human glioblastoma cells with recombinant HGF induces biologically relevant EGFR activation. EGFR phosphorylation at Tyr(845) and Tyr(1068) increased 6 to 24 h after cell stimulation with HGF and temporally coincided with the induction of transforming growth factor-alpha (~5-fold) and HB-EGF (~23-fold) expression. Tyr(845) and Tyr(1068) phosphorylation, in response to HGF, was inhibited by cycloheximide and actinomycin D, consistent with a requirement for DNA transcription and RNA translation. Specifically, blocking HB-EGF binding to EGFR with the antagonist CRM197 inhibited HGF-induced EGFR phosphorylation by 60% to 80% and inhibited HGF-induced S-G(2)-M transition. CRM197 also inhibited HGF-induced anchorage-dependent cell proliferation but had no effect on HGF-mediated cytoprotection. These findings establish that EGFR can be activated with functional consequences by HGF as a result of EGFR ligand expression. This transcription-dependent cross-talk between the HGF receptor c-Met and EGFR expands our understanding of receptor tyrosine kinase signaling networks and may have considerable consequences for oncogenic mechanisms and cancer therapeutics.

  20. Autologous Growth Factor Injections in Chronic Tendinopathy

    PubMed Central

    Sandrey, Michelle A.

    2014-01-01

    Reference: de Vos RJ, van Veldhoven PLJ, Moen MH, Weir A, Tol JL. Autologous growth factor injections in chronic tendinopathy: a systematic review. Br Med Bull. 2010;95:63–77. Clinical Question: The authors of this systematic review evaluated the literature to critically consider the effects of growth factors delivered through autologous whole-blood and platelet-rich–plasma (PRP) injections in managing wrist-flexor and -extensor tendinopathies, plantar fasciopathy, and patellar tendinopathy. The primary question was, according to the published literature, is there sufficient evidence to support the use of growth factors delivered through autologous whole-blood and PRP injections for chronic tendinopathy? Data Sources: The authors performed a comprehensive, systematic literature search in October 2009 using PubMed, MEDLINE, EMBASE, CINAHL, and the Cochrane library without time limits. The following key words were used in different combinations: tendinopathy, tendinosis, tendinitis, tendons, tennis elbow, plantar fasciitis, platelet rich plasma, platelet transfusion, and autologous blood or injection. The search was limited to human studies in English. All bibliographies from the initial literature search were also viewed to identify additional relevant studies. Study Selection: Studies were eligible based on the following criteria: (1) Articles were suitable (inclusion criteria) if the participants had been clinically diagnosed as having chronic tendinopathy; (2) the design had to be a prospective clinical study, randomized controlled trial, nonrandomized clinical trial, or prospective case series; (3) a well-described intervention in the form of a growth factor injection with either PRP or autologous whole blood was used; and (4) the outcome was reported in terms of pain or function (or both). Data Extraction: All titles and abstracts were assessed by 2 researchers, and all relevant articles were obtained. Two researchers independently read the full text of

  1. Expression and modulation of IL-1 alpha in murine keratinocytes

    SciTech Connect

    Ansel, J.C.; Luger, T.A.; Lowry, D.; Perry, P.; Roop, D.R.; Mountz, J.D.

    1988-04-01

    Murine and human keratinocytes produce an IL-1-like factor that appears to be similar if not identical to monocyte-derived IL-1. IL-1 may be an important mediator in cutaneous inflammatory responses, however, little is currently known concerning factors that may modulate IL-1 expression in keratinocytes. To address this issue we examined the effect of LPS, UV, and the cell differentiation state on murine keratinocyte IL-1 mRNA expression. Our results indicated that as with the murine P388D1 monocyte cell line, PAM 212 keratinocytes constitutively express abundant amounts of IL-1 alpha mRNA. On exposure to LPS (100 micrograms/ml) for 8 h there was more than 10 times the increase in PAM 212 IL-1 alpha mRNA which was accompanied by a sixfold increase in supernatant IL-1 activity. Similarly UV irradiation had a significant effect on keratinocyte IL-1 alpha expression. High dose UV (300 mJ/cm2) inhibited PAM 212 IL-1 alpha expression at 4, 8, 24, 48 h post-UV whereas a lower dose of UV (100 mJ/cm2) inhibited UV at 4 and 8 h post-UV, but induced IL-1 expression at 24 and 48 h post-UV. The expression of IL-1 alpha varied with the differentiation state of the keratinocytes. Freshly removed newborn murine keratinocytes were found to constitutively express IL-1 alpha mRNA. Keratinocytes grown in low (Ca2+) tissue culture media (0.05 mM) for 6 days, functionally and phenotypically become undifferentiated and express increased quantities of IL-1 alpha mRNA, whereas cells grown in high (Ca2+) media (1.2 mM) for 6 days become terminally differentiated and IL-1 expression ceased. Keratinocytes cultured for 3 days in low (Ca2+) conditions expressed an intermediate level of IL-1 alpha. In contrast, little or no IL-1 beta mRNA was detected in either the PAM 212 cells or newborn murine keratinocytes.

  2. Methylparaben potentiates UV-induced damage of skin keratinocytes.

    PubMed

    Handa, Osamu; Kokura, Satoshi; Adachi, Satoko; Takagi, Tomohisa; Naito, Yuji; Tanigawa, Toru; Yoshida, Norimasa; Yoshikawa, Toshikazu

    2006-10-01

    For many years, methylparaben (MP) has been used as a preservative in cosmetics. In this study, we investigated the effects of ultraviolet-B (UVB) exposure on MP-treated human skin keratinocytes. HaCaT keratinocyte was cultured in MP-containing medium for 24h, exposed to UVB (15 or 30 mJ/cm(2)) and further cultured for another 24h. Subsequent cellular viability was quantified by MTT-based assay and cell death was qualified by fluorescent microscopy and flow cytometry. Oxidative stress, nitric oxide (NO) production and cellular lipid peroxidation were measured using fluorescent probes. In addition, activation of nuclear factor kappa B and activator protein-1 was assessed by electro-mobility gel-shift assay. Practical concentrations of MP (0.003%) had a little or no effect on cellular viability, oxidative stress, NO production, lipid peroxidation and activation of nuclear transcription factors in HaCaT keratinocytes. Low-dose UVB also had little or no effect on these parameters in HaCaT keratinocytes. However, UVB exposure significantly increased cell death, oxidative stress, NO production, lipid peroxidation and activation of transcription factors in MP-treated HaCaT keratinocytes. These results indicate that MP, which has been considered a safe preservative in cosmetics, may have harmful effects on human skin when exposed to sunlight.

  3. Priming of mononuclear cells with a combination of growth factors enhances wound healing via high angiogenic and engraftment capabilities.

    PubMed

    Jin, Enze; Kim, Jong-Min; Kim, Sung-Whan

    2013-12-01

    Recently, we demonstrated that a specific combination of growth factors enhances the survival, adhesion and angiogenic potential of mononuclear cells (MNCs). In this study, we sought to investigate the changes of the angiogenic potential of MNCs after short-time priming with a specific combination of growth factors. MNCs were isolated using density gradient centrifugation and incubated with a priming cocktail containing epidermal growth factor (EGF), insulin-like growth factor (IGF)-1, fibroblast growth factor (FGF)-2, FMS-like tyrosine kinase (Flt)-3L , Angiopoietin (Ang)-1, granulocyte chemotactic protein (GCP)-2 and thrombopoietin (TPO) (all 400 ng/ml) for 15, 30 and 60 min. Wounds in nonobese diabetic-severe combined immune deficiency (NOD-SCID) mice were created by skin excision followed by cell transplantation. We performed a qRT-PCR analysis on the growth factor-primed cells. The angiogenic factors vascular endothelial growth factor (VEGF)-A, FGF-2, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF) and interleukin (IL)-8 and the anti-apoptotic factors IGF-1 and transforming growth factor-β1 were significantly elevated in the MNCs primed for 30 min. (T30) compared with the non-primed MNCs (T0). The scratch wound assay revealed that T30- conditioned media (CM) significantly increased the rate of fibroblast-mediated wound closure compared with the rates from T0-CM and human umbilical vein endothelial cells (HUVEC)-CM at 20 hrs. In vivo wound healing results revealed that the T30-treated wounds demonstrated accelerated wound healing at days 7 and 14 compared with those treated with T0. The histological analyses demonstrated that the number of engrafted cells and transdifferentiated keratinocytes in the wounds were significantly higher in the T30-transplanted group than in the T0-transplanted group. In conclusion, this study suggests that short-term priming of MNCs with growth factors might be alternative therapeutic option for cell

  4. Paracrine regulation of fibroblast aminopeptidase N/CD13 expression by keratinocyte-releasable stratifin.

    PubMed

    Lai, Amy; Ghaffari, Abdi; Li, Yunyuan; Ghahary, Aziz

    2011-12-01

    As wound healing proceeds into the tissue remodeling phase, cellular interactions become dominated by the interplay of keratinocytes with fibroblasts in the skin, which is largely mediated through paracrine signaling and greatly affects the molecular constitution of the extracellular matrix. We have recently identified aminopeptidase N (APN)/CD13 as a potential fibroblast receptor for 14-3-3 sigma (also known as stratifin), a keratinocyte-releasable protein with potent matrix metalloproteinase 1 (MMP1) stimulatory activity. The present study demonstrates that the expression of APN on dermal fibroblasts is regulated through paracrine signaling by keratinocyte-derived soluble factors. By using an in vitro keratinocyte-fibroblast co-culture system, we showed that APN expression in dermal fibroblasts is induced in the presence of keratinocytes or in response to keratinocyte-conditioned medium. Conditioned medium collected from differentiated keratinocytes further increases APN protein production, suggesting an amplified stimulatory effect by keratinocyte differentiation. Recombinant stratifin potently induces APN synthesis in a dose-dependent manner. A consistent correlation between the protein expression levels of APN and MMP1 was also observed. These results confirm paracrine regulation of APN expression in dermal fibroblasts by keratinocyte-derived stimuli, in particular stratifin, and provide evidence that APN may serve as a target in the regulation of MMP1 expression in epidermal-mesenchymal communication. PMID:21302309

  5. Systems Biology of Vascular Endothelial Growth Factors

    PubMed Central

    Mac Gabhann, Feilim; Popel, Aleksander S.

    2009-01-01

    Several cytokine families have roles in development, maintenance and remodeling of the microcirculation. Of these, the VEGF family is one of the best studied and one of the most complex. Five VEGF ligand genes and five cell surface receptor genes are known in the human, and each of these may be transcribed as multiple splice isoforms to generate an extensive family of proteins, many of which are subject to further proteolytic processing. Using the VEGF family as an example, we describe the current knowledge of growth factor expression, processing and transport in vivo. Experimental studies and computational simulations are being used to measure and predict the activity of these molecules, and we describe avenues of research that seek to fill the remaining gaps in our understanding of VEGF family behavior. PMID:18608994

  6. Modeling keratinocyte wound healing dynamics: Cell-cell adhesion promotes sustained collective migration.

    PubMed

    Nardini, John T; Chapnick, Douglas A; Liu, Xuedong; Bortz, David M

    2016-07-01

    The in vitro migration of keratinocyte cell sheets displays behavioral and biochemical similarities to the in vivo wound healing response of keratinocytes in animal model systems. In both cases, ligand-dependent Epidermal Growth Factor Receptor (EGFR) activation is sufficient to elicit collective cell migration into the wound. Previous mathematical modeling studies of in vitro wound healing assays assume that physical connections between cells have a hindering effect on cell migration, but biological literature suggests a more complicated story. By combining mathematical modeling and experimental observations of collectively migrating sheets of keratinocytes, we investigate the role of cell-cell adhesion during in vitro keratinocyte wound healing assays. We develop and compare two nonlinear diffusion models of the wound healing process in which cell-cell adhesion either hinders or promotes migration. Both models can accurately fit the leading edge propagation of cell sheets during wound healing when using a time-dependent rate of cell-cell adhesion strength. The model that assumes a positive role of cell-cell adhesion on migration, however, is robust to changes in the leading edge definition and yields a qualitatively accurate density profile. Using RNAi for the critical adherens junction protein, α-catenin, we demonstrate that cell sheets with wild type cell-cell adhesion expression maintain migration into the wound longer than cell sheets with decreased cell-cell adhesion expression, which fails to exhibit collective migration. Our modeling and experimental data thus suggest that cell-cell adhesion promotes sustained migration as cells pull neighboring cells into the wound during wound healing.

  7. Modeling keratinocyte wound healing dynamics: Cell-cell adhesion promotes sustained collective migration.

    PubMed

    Nardini, John T; Chapnick, Douglas A; Liu, Xuedong; Bortz, David M

    2016-07-01

    The in vitro migration of keratinocyte cell sheets displays behavioral and biochemical similarities to the in vivo wound healing response of keratinocytes in animal model systems. In both cases, ligand-dependent Epidermal Growth Factor Receptor (EGFR) activation is sufficient to elicit collective cell migration into the wound. Previous mathematical modeling studies of in vitro wound healing assays assume that physical connections between cells have a hindering effect on cell migration, but biological literature suggests a more complicated story. By combining mathematical modeling and experimental observations of collectively migrating sheets of keratinocytes, we investigate the role of cell-cell adhesion during in vitro keratinocyte wound healing assays. We develop and compare two nonlinear diffusion models of the wound healing process in which cell-cell adhesion either hinders or promotes migration. Both models can accurately fit the leading edge propagation of cell sheets during wound healing when using a time-dependent rate of cell-cell adhesion strength. The model that assumes a positive role of cell-cell adhesion on migration, however, is robust to changes in the leading edge definition and yields a qualitatively accurate density profile. Using RNAi for the critical adherens junction protein, α-catenin, we demonstrate that cell sheets with wild type cell-cell adhesion expression maintain migration into the wound longer than cell sheets with decreased cell-cell adhesion expression, which fails to exhibit collective migration. Our modeling and experimental data thus suggest that cell-cell adhesion promotes sustained migration as cells pull neighboring cells into the wound during wound healing. PMID:27105673

  8. Budesonide epimer R or dexamethasone selectively inhibit platelet-activating factor-induced or interleukin 1β-induced DNA binding activity of cis-acting transcription factors and cyclooxygenase-2 gene expression in human epidermal keratinocytes

    PubMed Central

    Lukiw, Walter J.; Pelaez, Ricardo Palacios; Martinez, Jorge; Bazan, Nicolas G.

    1998-01-01

    To further understand the molecular mechanism of glucocorticoid action on gene expression, DNA-binding activities of the cis-acting transcription factors activator protein 1 (AP1), AP2, Egr1 (zif268), NF-κB, the signal transducers and activators of transcription proteins gamma interferon activation site (GAS), Sis-inducible element, and the TATA binding protein transcription factor II D (TFIID) were examined in human epidermal keratinocytes. The cytokine interleukin 1β (IL-1β) and platelet-activating factor (PAF), both potent mediators of inflammation, were used as triggers for gene expression. Budesonide epimer R (BUDeR) and dexamethasone (DEX) were studied as potential antagonists. BUDeR or DEX before IL-1β- or PAF-mediated gene induction elicited strong inhibition of AP1-, GAS-, and in particular NF-κB-DNA binding (P < 0.001, ANOVA). Only small effects were noted on AP2, Egr1 (zif268), and Sis-inducible element-DNA binding (P > 0.05). No significant effect was noted on the basal transcription factor TFIID recognition of TATA-containing core promoter sequences (P > 0.68). To test the hypothesis that changing cis-acting transcription factor binding activity may be involved in inflammatory-response related gene transcription, RNA message abundance for human cyclooxygenase (COX)-1 and -2 (E.C.1.14.99.1) was assessed in parallel by using reverse transcription–PCR. Although the COX-1 gene was found to be expressed at constitutively low levels, the TATA-containing COX-2 gene, which contains AP1-like, GAS, and NF-κB DNA-binding sites in its immediate promoter, was found to be strongly induced by IL-1β or PAF (P < 0.001). BUDeR and DEX both suppressed COX-2 RNA message generation; however, no correlation was associated with TFIID–DNA binding. These results suggest that on stimulation by mediators of inflammation, although the basal transcription machinery remains intact, modulation of cis-activating transcription factor AP1, GAS, and NF-κB-DNA binding by the

  9. Epidermal growth factor (urogastrone) in human tissues.

    PubMed

    Hirata, Y; Orth, D N

    1979-04-01

    Human epidermal growth factor (hEGF), which stimulates the growth of a variety of tissues, was first isolated from mouse submandibular glands, but is also excreted in large amounts (about 50 micrograms/day) in human urine and is probably identical to human beta-urogastrone (hUG), a potent inhibitor of stimulated gastric acid secretion. However, the primary tissue source of hEGF/hUG is as yet unknown. The hEGF/hUG in homogenates of human salivary glands and a wide variety of other endocrine and nonendocrine tissues was extracted by Amberlite CG-50 cation exchange chromatography and immune affinity chromatography using the immunoglobulin fraction of rabbit anti-hEGF serum covalently bound to agarose. The extracts were subjected to homologous hEGF RIA. Immunoreactive hEGF was found in extracts of adult submandibular gland, thyroid gland, duodenum, jejunum, and kidney, but not in several fetal tissues. The tissue immunoreactive hEGF was similar to standard hEGF in terms of immunoreactivity and elution from Sephadex G-50 Fine resin, but its concentrations were very low (1.3-5.5 ng/g wet tissue). Thus, it is not certain that these tissues represent the only source of the large amounts of hEGF/hUG that appear to be filtered by the kidneys each day.

  10. The Fibroblast Growth Factor signaling pathway

    PubMed Central

    Ornitz, David M; Itoh, Nobuyuki

    2015-01-01

    The signaling component of the mammalian Fibroblast Growth Factor (FGF) family is comprised of eighteen secreted proteins that interact with four signaling tyrosine kinase FGF receptors (FGFRs). Interaction of FGF ligands with their signaling receptors is regulated by protein or proteoglycan cofactors and by extracellular binding proteins. Activated FGFRs phosphorylate specific tyrosine residues that mediate interaction with cytosolic adaptor proteins and the RAS-MAPK, PI3K-AKT, PLCγ, and STAT intracellular signaling pathways. Four structurally related intracellular non-signaling FGFs interact with and regulate the family of voltage gated sodium channels. Members of the FGF family function in the earliest stages of embryonic development and during organogenesis to maintain progenitor cells and mediate their growth, differentiation, survival, and patterning. FGFs also have roles in adult tissues where they mediate metabolic functions, tissue repair, and regeneration, often by reactivating developmental signaling pathways. Consistent with the presence of FGFs in almost all tissues and organs, aberrant activity of the pathway is associated with developmental defects that disrupt organogenesis, impair the response to injury, and result in metabolic disorders, and cancer. © 2015 Wiley Periodicals, Inc. PMID:25772309

  11. [Growth Hormone-Insulin Growth Factor I (GH-IGF-I) axis and growth].

    PubMed

    Castell, A-L; Sadoul, J-L; Bouvattier, C

    2013-10-01

    Normal human linear growth results from an evolutionary process expressing the sum effect of multiple genes. The growth hormone (GH) - insulin like growth factor (IGF)-I axis is one of the main actors in the growth process. Defects in this axis can be responsible for short or tall stature. Short stature is defined as smaller than - 2 standard deviations (SD). It is a very common reason for consultation in pediatrics; indeed, 2.5 % of children are concerned. Multiple causes make diagnosis difficult. In this article, we detail the most common constitutional causes of small size, including those related to a defect in the GH-IGF-I axis. Then, we report, the first results of the clinical and genetic study conducted on 213 patients with gigantism. Tall stature is defined by a height superior to 2 SD. Finally, recent work linking epigenetics and growth - via signaling pathways of GH-IGF-I axis - will be presented. PMID:24356290

  12. [Growth Hormone-Insulin Growth Factor I (GH-IGF-I) axis and growth].

    PubMed

    Castell, A-L; Sadoul, J-L; Bouvattier, C

    2013-10-01

    Normal human linear growth results from an evolutionary process expressing the sum effect of multiple genes. The growth hormone (GH) - insulin like growth factor (IGF)-I axis is one of the main actors in the growth process. Defects in this axis can be responsible for short or tall stature. Short stature is defined as smaller than - 2 standard deviations (SD). It is a very common reason for consultation in pediatrics; indeed, 2.5 % of children are concerned. Multiple causes make diagnosis difficult. In this article, we detail the most common constitutional causes of small size, including those related to a defect in the GH-IGF-I axis. Then, we report, the first results of the clinical and genetic study conducted on 213 patients with gigantism. Tall stature is defined by a height superior to 2 SD. Finally, recent work linking epigenetics and growth - via signaling pathways of GH-IGF-I axis - will be presented.

  13. The role of the tetraspanin CD151 in primary keratinocyte and fibroblast functions: Implications for wound healing

    SciTech Connect

    Geary, Sean M.; Cowin, Allison J.; Copeland, Ben; Baleato, Rosa M.; Miyazaki, Kaoru; Ashman, Leonie K.

    2008-07-01

    Previous studies showed that CD151-null mice have a skin wound healing deficit. To gain an understanding of the role of CD151 in re-epithelialisation and dermal contraction, keratinocyte and fibroblast functions were assayed. Primary CD151-null keratinocytes displayed defective migration on Matrigel (a basement membrane equivalent) and laminin-332, the primary adhesion component of basement membranes, but not on collagen-I. Adhesion, spreading and proliferation were also deficient on laminin-332, but not collagen-I. The data suggest that loss of CD151 impairs the function of its primary interaction partners, integrin {alpha}3{beta}1- and/or {alpha}6{beta}4 which bind to laminin-332. Skin fibroblasts also produce CD151 mRNA. CD151-null fibroblasts migrated significantly faster on collagen I than wild type fibroblasts, confirming that they possess functional collagen receptors. However, no significant decrease in the ability of CD151-null fibroblasts to cause contraction in floating collagen gel assays in response to transforming growth factor beta-1 (TGF-{beta}1) or platelet derived growth factor (PDGF-BB) was observed, nor was there an effect on fibroblast adhesion or proliferation on collagen-I. The data implicate CD151 as a facilitator of laminin-332-mediated keratinocyte functions that impact on the re-epithelialisation process intrinsic to wound healing and further suggest a potential novel role for CD151 in fibroblast migration.

  14. Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery.

    PubMed

    Wing Moon Lam, Raymond; Abbah, Sunny Akogwu; Ming, Wang; Naidu, Mathanapriya; Ng, Felly; Tao, Hu; Goh Cho Hong, James; Ting, Kang; Hee Kit, Wong

    2016-01-01

    During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries. PMID:27585207

  15. Transforming growth factor-β and fibrosis

    PubMed Central

    Verrecchia, Franck; Mauviel, Alain

    2007-01-01

    Transforming growth factor-β (TGF-β), a prototype of multifunctional cytokine, is a key regulator of extracellular matrix (ECM) assembly and remodeling. Specifically, TGF-β isoforms have the ability to induce the expression of ECM proteins in mesenchymal cells, and to stimulate the production of protease inhibitors that prevent enzymatic breakdown of the ECM. Elevated TGF-β expression in affected organs, and subsequent deregulation of TGF-β functions, correlates with the abnormal connective tissue deposition observed during the onset of fibrotic diseases. During the last few years, tremendous progress has been made in the understanding of the molecular aspects of intracellular signaling downstream of the TGF-β receptors. In particular, Smad proteins, TGF-β receptor kinase substrates that translocate into the cell nucleus to act as transcription factors, have been studied extensively. The role of Smad3 in the transcriptional regulation of typeIcollagen gene expression and in the development of fibrosis, demonstrated both in vitro and in animal models with a targeted deletion of Smad3, is of critical importance because it may lead to novel therapeutic strategies against these diseases. This review focuses on the mechanisms underlying Smad modulation of fibrillar collagen expression and how it relates to fibrotic processes. PMID:17589920

  16. Design of Growth Factor Sequestering Biomaterials

    PubMed Central

    Belair, David G.; Le, Ngoc Nhi; Murphy, William L.

    2014-01-01

    Growth factors (GFs) are major regulatory proteins that can govern cell fate, migration, and organization. Numerous aspects of the cell milieu can modulate cell responses to GFs, and GF regulation is often achieved by the native extracellular matrix (ECM). For example, the ECM can sequester GFs and thereby control GF bioavailability. In addition, GFs can exert distinct effects depending on whether they are sequestered in solution, at two-dimensional interfaces, or within three-dimensional matrices. Understanding how the context of GF sequestering impacts cell function in the native ECM can instruct the design of soluble or insoluble GF sequestering moieties, which can then be used in a variety of bioengineering applications. This Feature Article provides an overview of the natural mechanisms of GF sequestering in the cell milieu, and reviews the recent bioengineering approaches that have sequestered GFs to modulate cell function. Results to date demonstrate that the cell response to GF sequestering depends on the affinity of the sequestering interaction, the spatial proximity of sequestering in relation to cells, the source of the GF (supplemented or endogenous), and the phase of the sequestering moiety (soluble or insoluble). We highlight the importance of context for the future design of biomaterials that can leverage endogenous molecules in the cell milieu and mitigate the need for supplemented factors. PMID:25182455

  17. Proliferation of normal human keratinocytes on silicone substrates.

    PubMed

    Rosdy, M; Grisoni, B; Clauss, L C

    1991-07-01

    Several polydimethylsiloxane elastomers and gels were tested as culture substrates for proliferating normal human epidermal keratinocytes. Growth kinetics of normal human keratinocytes (NHK) and dermal fibroblasts were compared on 'very soft', 'soft' and 'hard' silicone gels, as well as on standard cell culture polystyrene dishes. Water contact angles and chemical compositions (IRFT-HATR) of the different silicone surfaces were found to be equivalent, although very different from standard cell culture polystyrene. The topography of the surfaces as well as the shape of the keratinocytes and fibroblasts grown on the different substrates were visualized by scanning electron microscopy, and compared. Although the surface softness and topography of the substrates differed markedly, dermal fibroblasts proliferated in serum-containing medium in equivalent manner on all substrates. Again no correlation could be found between the characteristics and the attachment of the substrates and rapid proliferation of the epidermal keratinocytes in defined medium. The epidermal keratinocytes spread, secreted a structured extracellular matrix network and grew up to confluence on all silicone substrates (elastomers and gels), except the relatively 'hard' silicone gel; this could be due to a direct interference by the waves observed on the silicone gel surfaces. PMID:1654138

  18. Effect of Recombinant Human Keratinocyte Growth Factor (rHuKGF, Palifermin) on Radiation-Induced Mouse Urinary Bladder Dysfunction

    SciTech Connect

    Jaal, Jana Doerr, Wolfgang

    2007-10-01

    Purpose: To determine the effect of Palifermin (rHuKGF) on acute and late radiation effects in mouse urinary bladder. Methods and Materials: Graded radiation doses were applied on day 0. Single subcutaneous injections of Palifermin (15 mg/kg) were given on day -2 or day +2. Changes in bladder function (i.e., a reduction in bladder volume by {>=}50% of the individual preirradiation value) were assessed by cystometry. Results: Early changes in mouse bladder after irradiation occur in two phases. In the first early phase, a single injection of Palifermin on day -2 increased the ED{sub 50} (dose associated with a positive bladder response in 50% of the mice) from 20.0 {+-} 3.3 Gy to 27.1 {+-} 6.9 Gy (p < .0051). Palifermin given on day +2 was not beneficial. No significant effects of Palifermin were seen in the second early phase. However, Palifermin administration before, but not after, irradiation, also modified late radiation effects, with an ED{sub 50} of 22.2 {+-} 4.8 Gy compared with 16.2 {+-} 4.9 Gy in control animals (p < .0187). Conclusions: Initial early functional changes in the mouse urinary bladder after irradiation as well as late effects can be significantly reduced by a single administration of Palifermin before irradiation.

  19. Human Keratinocytes Are Vanilloid Resistant

    PubMed Central

    Pecze, László; Szabó, Kornélia; Széll, Márta; Jósvay, Katalin; Kaszás, Krisztián; Kúsz, Erzsébet; Letoha, Tamás; Prorok, János; Koncz, István; Tóth, András; Kemény, Lajos; Vizler, Csaba; Oláh, Zoltán

    2008-01-01

    Background Use of capsaicin or resiniferatoxin (RTX) as analgesics is an attractive therapeutic option. RTX opens the cation channel inflammatory pain/vanilloid receptor type 1 (TRPV1) permanently and selectively removes nociceptive neurons by Ca2+-cytotoxicity. Paradoxically, not only nociceptors, but non-neuronal cells, including keratinocytes express full length TRPV1 mRNA, while patient dogs and experimental animals that underwent topical treatment or anatomically targeted molecular surgery have shown neither obvious behavioral, nor pathological side effects. Methods To address this paradox, we assessed the vanilloid sensitivity of the HaCaT human keratinocyte cell line and primary keratinocytes from skin biopsies. Results Although both cell types express TRPV1 mRNA, neither responded to vanilloids with Ca2+-cytotoxicity. Only ectopic overproduction of TRPV1 rendered HaCaT cells sensitive to low doses (1–50 nM) of vanilloids. The TRPV1-mediated and non-receptor specific Ca2+-cytotoxity ([RTX]>15 µM) could clearly be distinguished, thus keratinocytes were indeed resistant to vanilloid-induced, TRPV1-mediated Ca2+-entry. Having a wider therapeutic window than capsaicin, RTX was effective in subnanomolar range, but even micromolar concentrations could not kill human keratinocytes. Keratinocytes showed orders of magnitudes lower TRPV1 mRNA level than sensory ganglions, the bona fide therapeutic targets in human pain management. In addition to TRPV1, TRPV1b, a dominant negative splice variant was also noted in keratinocytes. Conclusion TRPV1B expression, together with low TRPV1 expression, may explain the vanilloid paradox: even genuinely TRPV1 mRNA positive cells can be spared with therapeutic (up to micromolar) doses of RTX. This additional safety information might be useful for planning future human clinical trials. PMID:18852901

  20. Agent Based Modelling Helps in Understanding the Rules by Which Fibroblasts Support Keratinocyte Colony Formation

    PubMed Central

    Sun, Tao; McMinn, Phil; Holcombe, Mike; Smallwood, Rod; MacNeil, Sheila

    2008-01-01

    Background Autologous keratincoytes are routinely expanded using irradiated mouse fibroblasts and bovine serum for clinical use. With growing concerns about the safety of these xenobiotic materials, it is desirable to culture keratinocytes in media without animal derived products. An improved understanding of epithelial/mesenchymal interactions could assist in this. Methodology/Principal Findings A keratincyte/fibroblast o-culture model was developed by extending an agent-based keratinocyte colony formation model to include the response of keratinocytes to both fibroblasts and serum. The model was validated by comparison of the in virtuo and in vitro multicellular behaviour of keratinocytes and fibroblasts in single and co-culture in Greens medium. To test the robustness of the model, several properties of the fibroblasts were changed to investigate their influence on the multicellular morphogenesis of keratinocyes and fibroblasts. The model was then used to generate hypotheses to explore the interactions of both proliferative and growth arrested fibroblasts with keratinocytes. The key predictions arising from the model which were confirmed by in vitro experiments were that 1) the ratio of fibroblasts to keratinocytes would critically influence keratinocyte colony expansion, 2) this ratio needed to be optimum at the beginning of the co-culture, 3) proliferative fibroblasts would be more effective than irradiated cells in expanding keratinocytes and 4) in the presence of an adequate number of fibroblasts, keratinocyte expansion would be independent of serum. Conclusions A closely associated computational and biological approach is a powerful tool for understanding complex biological systems such as the interactions between keratinocytes and fibroblasts. The key outcome of this study is the finding that the early addition of a critical ratio of proliferative fibroblasts can give rapid keratinocyte expansion without the use of irradiated mouse fibroblasts and bovine

  1. Growth factors and cardiovascular structure. Implications for calcium antagonist therapy.

    PubMed

    Re, R N; Chen, L

    1991-07-01

    Abnormalities of cellular growth regulation are integral to the development of cardiovascular disorders such as atherogenesis, ventricular hypertrophy, and diabetic glomerulopathy. Moreover, cellular growth is in large measure controlled by peptide and nonpeptide growth factors that mediate their actions, in part, through the transcriptional regulation of normal cellular genes called protooncogenes. Because angiotensin II is one such growth regulatory factor and because changes in intracellular calcium are intimately involved in the action of angiotensin and other growth factors, it is likely that inhibitors of angiotensin action and calcium-channel-blocking agents will be found to have useful growth regulatory properties. PMID:1910639

  2. Concentration of Fibrin and Presence of Plasminogen Affect Proliferation, Fibrinolytic Activity, and Morphology of Human Fibroblasts and Keratinocytes in 3D Fibrin Constructs

    PubMed Central

    Reinertsen, Erik; Skinner, Michael; Wu, Benjamin

    2014-01-01

    Fibrin is a hemostatic protein found in the clotting cascade. It is used in the operating room to stop bleeding and deliver cells and growth factors to heal wounds. However, formulations of clinically approved fibrin are optimized for hemostasis, and the extent to which biochemical and physical cues in fibrin mediate skin cell behavior is not fully understood nor utilized in the design of biomaterials. To determine if the concentration of fibrinogen and the presence of plasminogen affect cell behavior relevant to wound healing, we fabricated three-dimensional fibrin constructs made from 5, 10, or 20 mg/mL of clinical fibrin or plasminogen-depleted (PD) fibrin. We cultured dermal fibroblasts or epidermal keratinocytes in these constructs. Fibroblasts proliferated similarly in both types of fibrin, but keratinocytes proliferated more in low concentrations of clinical fibrin and less in PD fibrin. Clinical fibrin constructs with fibroblasts were less stiff and degraded faster than PD fibrin constructs with fibroblasts. Similarly, keratinocytes degraded clinical fibrin, but not PD fibrin. Fibroblast spreading varied with fibrin concentration in both types of fibrin. In conclusion, the concentration of fibrinogen and the presence of plasminogen affect fibroblast and keratinocyte proliferation, morphology, and fibrin degradation. Creating materials with heterogeneous regions of fibrin formulations and concentrations could be a novel strategy for controlling the phenotype of encapsulated fibroblasts and keratinocytes, and the subsequent biomechanical properties of the construct. However, other well-investigated aspects of wound healing remain to be utilized in the design of fibrin biomaterials, such as autocrine and paracrine signaling between fibroblasts, keratinocytes, and immune cells. PMID:24738616

  3. Proteolytic and non-proteolytic activation of keratinocyte-derived latent TGF-β1 induces fibroblast differentiation in a wound-healing model using rat skin.

    PubMed

    Hata, Shozaburo; Okamura, Kazuhiko; Hatta, Mitsutoki; Ishikawa, Hiroyuki; Yamazaki, Jun

    2014-01-01

    Transforming growth factor-β1 (TGF-β1) reportedly causes the differentiation of fibroblasts to myofibroblasts during wound healing. We investigated the mechanism underlying the activation of latent TGF-β1 released by keratinocytes in efforts to identify promising pharmacological approaches for the prevention of hypertrophic scar formation. A three-dimensional collagen gel matrix culture was prepared using rat keratinocytes and dermal fibroblasts. Stratified keratinocytes promoted the TGF receptor-dependent increase in α-smooth muscle actin (α-SMA) immunostaining and mRNA levels in fibroblasts. Latent TGF-β1 was found to be localized suprabasally and secreted. α-SMA expression was inhibited by an anti-αv-integrin antibody and a matrix metalloproteinase (MMP) inhibitor, GM6001. In a two-dimensional fibroblast culture, α-SMA expression depended on the production of endogenous TGF-β1 and required αv-integrin or MMP for the response to recombinant latent TGF-β1. In keratinocyte-conditioned medium, MMP-dependent latent TGF-β1 secretion was detected. Applying this medium to the fibroblast culture enhanced α-SMA production. This effect was decreased by GM6001, the anti-αv-integrin antibody, or the preabsorption of latent TGF-β1. These results indicate that keratinocytes secrete latent TGF-β1, which is liberated to fibroblasts over distance and is activated to produce α-SMA with the aid of a positive-feedback loop. MMP inhibition was effective for targeting both keratinocytes and fibroblasts in this model. PMID:24492413

  4. Transcriptional down-regulation of epidermal growth factor receptors by nerve growth factor treatment of PC12 cells.

    PubMed

    Shibutani, M; Lazarovici, P; Johnson, A C; Katagiri, Y; Guroff, G

    1998-03-20

    Treatment of PC12 cells with nerve growth factor leads to a decrease in the number of epidermal growth factor receptors on the cell membrane. The mRNA for the epidermal growth factor receptor decreases in a comparable fashion. This decrease appears due to a decrease in the transcription of the epidermal growth factor receptor gene because first, there is no difference in the stability of the epidermal growth factor receptor mRNA, second, newly transcribed epidermal growth factor receptor mRNA is decreased in nerve growth factor-differentiated cells, and third, constructs containing the promoter region of the epidermal growth factor receptor gene are transcribed much less readily in nerve growth factor-differentiated cells than in untreated cells. The decreases in mRNA are not seen in the p140(trk)-deficient variant PC12nnr5 cells nor in cells containing either dominant-negative Ras or dominant-negative Src. Treatment with nerve growth factor also increases the cellular content of GCF2, a putative transcription factor inhibitory for the transcription of the epidermal growth factor receptor gene. The increase in GCF2, like the decrease in the epidermal growth factor receptor mRNA, is not seen in PC12nnr5 cells nor in cells expressing either dominant-negative Ras or dominant-negative Src. The results suggest that nerve growth factor-induced down-regulation of the epidermal growth factor receptor is under transcriptional control, is p140(trk)-, Ras-, and Src-dependent, and may involve transcriptional repression by GCF2.

  5. Endorsement of Growth Factors in Experiential Training Groups

    ERIC Educational Resources Information Center

    Kiweewa, John; Gilbride, Dennis; Luke, Melissa; Seward, Derek

    2013-01-01

    The purpose of this study was to identify student growth factors during a semester long Master's level group counseling class. Results indicated that 12 growth factors accounted for 86% of the total number of critical incidents that participants reported as influencing their personal growth and awareness during the group experience. Two other…

  6. Gene Expression of Growth Factors and Growth Factor Receptors for Potential Targeted Therapy of Canine Hepatocellular Carcinoma

    PubMed Central

    IIDA, Gentoku; ASANO, Kazushi; SEKI, Mamiko; SAKAI, Manabu; KUTARA, Kenji; ISHIGAKI, Kumiko; KAGAWA, Yumiko; YOSHIDA, Orie; TESHIMA, Kenji; EDAMURA, Kazuya; WATARI, Toshihiro

    2013-01-01

    ABSTRACT The purpose of this study was to evaluate the gene expression of growth factors and growth factor receptors of primary hepatic masses, including hepatocellular carcinoma (HCC) and nodular hyperplasia (NH), in dogs. Quantitative real-time reverse transcriptase-polymerase chain reaction was performed to measure the expression of 18 genes in 18 HCCs, 10 NHs, 11 surrounding non-cancerous liver tissues and 4 healthy control liver tissues. Platelet-derived growth factor-B (PDGF-B), transforming growth factor-α, epidermal growth factor receptor, epidermal growth factor and hepatocyte growth factor were found to be differentially expressed in HCC compared with NH and the surrounding non-cancerous and healthy control liver tissues. PDGF-B is suggested to have the potential to become a valuable ancillary target for the treatment of canine HCC. PMID:24189579

  7. Growth factor involvement in tension-induced skeletal muscle growth

    NASA Technical Reports Server (NTRS)

    Vandenburgh, Herman W.

    1987-01-01

    New muscle tissue culture techniques were developed to grow embryonic skeletal myofibers which are able to differentiate into more adultlike myofibers. Studies on mechanical simulation of cultured muscle cell growth will now be more directly applicable to mechanically-induced growth in adult muscle, and lead to better models for understanding muscle tissue atrophy caused by disuse in the microgravity of space.

  8. [Stem cells and growth factors in wound healing].

    PubMed

    Pikuła, Michał; Langa, Paulina; Kosikowska, Paulina; Trzonkowski, Piotr

    2015-01-02

    Wound healing is a complex process which depends on the presence of various types of cells, growth factors, cytokines and the elements of extracellular matrix. A wound is a portal of entry for numerous pathogens, therefore during the evolution wound healing process has formed very early, being critical for the survival of every individual. Stem cells, which give rise to their early descendants progenitor cells and subsequently differentiated cells, play a specific role in the process of wound healing. Among the most important cells which take part in wound healing the following cells need to be distinguished: epidermal stem cells, dermal precursor of fibroblasts, adipose-derived stem cells as well as bone marrow cells. The activity of these cells is strictly regulated by various growth factors, inter alia epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF), vascular endothelial growth factor (VEGF). Any disorders in functioning of stem cells and biological activity of growth factors may lead to the defects in wound healing, for instance delayed wound healing or creation of hypertrophic scars. Therefore, knowledge concerning the mechanisms of wound healing is extremely essential from clinical point of view. In this review the current state of the knowledge of the role of stem cells and growth factors in the process of wound healing has been presented. Moreover, some clinical aspects of wound healing as well as the possibility of the therapy based on stem cells and growth factors have included.

  9. Single Low-Dose Radiation Induced Regulation of Keratinocyte Differentiation in Calcium-Induced HaCaT Cells

    PubMed Central

    Hahn, Hyung Jin; Youn, Hae Jeong; Cha, Hwa Jun; Kim, Karam; An, Sungkwan

    2016-01-01

    Background We are continually exposed to low-dose radiation (LDR) in the range 0.1 Gy from natural sources, medical devices, nuclear energy plants, and other industrial sources of ionizing radiation. There are three models for the biological mechanism of LDR: the linear no-threshold model, the hormetic model, and the threshold model. Objective We used keratinocytes as a model system to investigate the molecular genetic effects of LDR on epidermal cell differentiation. Methods To identify keratinocyte differentiation, we performed western blots using a specific antibody for involucrin, which is a precursor protein of the keratinocyte cornified envelope and a marker for keratinocyte terminal differentiation. We also performed quantitative polymerase chain reaction. We examined whether LDR induces changes in involucrin messenger RNA (mRNA) and protein levels in calcium-induced keratinocyte differentiation. Results Exposure of HaCaT cells to LDR (0.1 Gy) induced p21 expression. p21 is a key regulator that induces growth arrest and represses stemness, which accelerates keratinocyte differentiation. We correlated involucrin expression with keratinocyte differentiation, and examined the effects of LDR on involucrin levels and keratinocyte development. LDR significantly increased involucrin mRNA and protein levels during calcium-induced keratinocyte differentiation. Conclusion These studies provide new evidence for the biological role of LDR, and identify the potential to utilize LDR to regulate or induce keratinocyte differentiation. PMID:27489424

  10. Human melanocytes mitigate keratinocyte-dependent contraction in an in vitro collagen contraction assay.

    PubMed

    Rakar, Jonathan; Krammer, Markus P; Kratz, Gunnar

    2015-08-01

    Scarring is an extensive problem in burn care, and treatment can be especially complicated in cases of hypertrophic scarring. Contraction is an important factor in scarring but the contribution of different cell types remains unclear. We have investigated the contractile behavior of keratinocytes, melanocytes and fibroblasts by using an in vitro collagen gel assay aimed at identifying a modulating role of melanocytes in keratinocyte-mediated contraction. Cells were seeded on a collagen type I gel substrate and the change in gel dimensions were measured over time. Hematoxylin & Eosin-staining and immunohistochemistry against pan-cytokeratin and microphthalmia-associated transcription factor showed that melanocytes integrated between keratinocytes and remained there throughout the experiments. Keratinocyte- and fibroblast-seeded gels contracted significantly over time, whereas melanocyte-seeded gels did not. Co-culture assays showed that melanocytes mitigate the keratinocyte-dependent contraction (significantly slower and 18-32% less). Fibroblasts augmented the contraction in most assays (approximately 6% more). Non-contact co-cultures showed some influence on the keratinocyte-dependent contraction. Results show that mechanisms attributable to melanocytes, but not fibroblasts, can mitigate keratinocyte contractile behavior. Contact-dependent mechanisms are stronger modulators than non-contact dependent mechanisms, but both modes carry significance to the contraction modulation of keratinocytes. Further investigations are required to determine the mechanisms involved and to determine the utility of melanocytes beyond hypopigmentation in improved clinical regimes of burn wounds and wound healing.

  11. Transcriptional modulation of transin gene expression by epidermal growth factor and transforming growth factor beta

    SciTech Connect

    Machida, C.M.; Muldoon, L.L.; Rodland, K.D.; Magun, B.E.

    1988-06-01

    Transin is a transformation-associated gene which is expressed constitutively in rat fibroblasts transformed by a variety of oncogenes and in malignant mouse skin carcinomas but not benign papillomas or normal skin. It has been demonstrated that, in nontransformed Rat-1 cells, transin RNA expression is modulated positively by epidermal growth factor (EGF) and negatively by transforming growth factor beta (TGF-BETA); other peptide growth factors were found to have no effect on transin expression. Results presented here indicate that both protein synthesis and continuous occupancy of the EGF receptor by EGF were required for sustained induction of transin RNA. Treatment with TGF-BETA inhibited the ability of EGF to induce transin, whether assayed at the transcriptional level by nuclear run-on analysis or at the level of transin RNA accumulation by Northern (RNA) blot analysis of cellular RNA. TGF-BETA both blocked initial production of transin transcription by EGF and halted established production of transin transcripts during prolonged treatment. These results suggest that TGF-BETA acts at the transcriptional level to antagonize EGF-mediated induction of transin gene expression.

  12. Direct binding of hepatocyte growth factor and vascular endothelial growth factor to CD44v6

    PubMed Central

    Volz, Yvonne; Koschut, David; Matzke-Ogi, Alexandra; Dietz, Marina S.; Karathanasis, Christos; Richert, Ludovic; Wagner, Moritz G.; Mély, Yves; Heilemann, Mike; Niemann, Hartmut H.; Orian-Rousseau, Véronique

    2015-01-01

    CD44v6, a member of the CD44 family of transmembrane glycoproteins is a co-receptor for two receptor tyrosine kinases (RTKs), Met and VEGFR-2 (vascular endothelial growth factor receptor 2). CD44v6 is not only required for the activation of these RTKs but also for signalling. In order to understand the role of CD44v6 in Met and VEGFR-2 activation and signalling we tested whether CD44v6 binds to their ligands, HGF (hepatocyte growth factor) and VEGF (vascular endothelial growth factor), respectively. FACS analysis and cellular ELISA showed binding of HGF and VEGF only to cells expressing CD44v6. Direct binding of CD44v6 to HGF and VEGF was demonstrated in pull-down assays and the binding affinities were determined using MicroScale Thermophoresis, fluorescence correlation spectroscopy and fluorescence anisotropy. The binding affinity of CD44v6 to HGF is in the micromolar range in contrast with the high-affinity binding measured in the case of VEGF and CD44v6, which is in the nanomolar range. These data reveal a heparan sulfate-independent direct binding of CD44v6 to the ligands of Met and VEGFR-2 and suggest different roles of CD44v6 for these RTKs. PMID:26181364

  13. Epidermal growth factor receptors in the oesophagus.

    PubMed Central

    Jankowski, J; Murphy, S; Coghill, G; Grant, A; Wormsley, K G; Sanders, D S; Kerr, M; Hopwood, D

    1992-01-01

    The quantity and distribution of epidermal growth factor receptors (EGF-R) in oesophageal mucosa was studied in the oesophagus in order to determine its role in oesophageal disease. Fifty five biopsies were taken from different levels of the oesophagus in 25 consecutive patients undergoing endoscopy. Another group of eight patients with histologically proven Barrett's oesophagitis had a biopsy taken from the area of columnar lined oesophagus. A peripheral, membranous pattern was seen predominantly confined to the basal and immediately suprabasal cells in all of the first group of patients. In the superficial cells a few granular cytoplasmic structures were positive. All patients with Barrett's oesophagitis showed EGF-R staining of the surface epithelium. A computerised planimeter was used to determine the proportion of stained areas of squamous cells which were expressed as a percentage of the total area of squamous cells. The difference in the area of cells stained for EGF-R between normal and inflamed oesophageal mucosa (29.5% and 43.1% respectively) was significant (p less than 0.001). Images Figure 1 PMID:1582583

  14. Nerve growth factor actions on the brain

    SciTech Connect

    Martinez, H.J.

    1989-01-01

    We examined the effect of the trophic protein, nerve growth factor (NGF), on cultures of fetal rat neostriatum and basal forebrain-medial septal area (BF-MS) to define its role in brain development. Treatment of cultures with NGF resulted in an increase in the specific activity of the cholinergic enzyme choline acetyltransferase (CAT) in both brain areas. CAT was immunocytochemically localized to neurons. In the BF-MS, NGF treatment elicited a marked increase in staining intensity and an apparent increase in the number of CAT-positive neurons. Moreover, treatment of BF-MS cultures with NGF increased the activity of acetylcholinesterase, suggesting that the cholinergic neuron as a whole was affected. To begin defining mechanisms of action of NGF in the BF-MS, we detected NGF receptors by two independent methods. Receptors were localized to two different cellular populations: neuron-like cells, and non-neuron-like cells. Dissociation studies with ({sup 125}I)NGF suggested that high affinity receptors were localized to the neuron-like population. Only low-affinity receptors were localized to the non-neuron-like cells. Moreover, employing combined immunocytochemistry and ({sup 125}I)NGF autoradiography, we detected a subpopulation of CAT-containing neutrons that exhibited high-affinity binding. Unexpectedly, a gamma-aminobutyric acid (GABA)-containing cell group also expressed high affinity binding. However, only subsets of cholinergic or GABA neurons expressed high-affinity biding, suggesting that these transmitter populations are composed of differentially response subpopulations.

  15. [Epidermal growth factor, innovation and safety].

    PubMed

    Esquirol Caussa, Jordi; Herrero Vila, Elisabeth

    2015-10-01

    Bioidentical recombinant human epidermal growth factor (rhEGF) is available in concentrations and purity suitable for therapeutic use in long time stable formulations. Beneficial effects in several skin pathologies and lesions have been reported (traumatic and surgical wound healing, laser induced wounds, abnormal scars, keloids, radiation or chemotherapy induced dermatitis, post inflammatory hyperpigmentation or for skin aging damage repairing) and also may be considered for the treatment of several oropharingeal and high gastroesophageal tract mucosa diseases (mouth sores, pharyngeal fistulas, ulcers), and several corneal or conjunctive mucosa lesions. rhEGF has not shown any important side or collateral effects in humans or in laboratory experimentation animals, showing optimal tolerability and safety with continuous use for months. Compounding gives advantages of versatility, individualization, personalization, molecular stability, safety and effectiveness in ideal conditions, showing good tissue penetration, both on intact skin and skin lesions that expose the lower planes to the surface. rhEGF compounds can be considered for prevention or as a treatment of diverse skin and mucosa diseases and conditions through compounding preparations.

  16. Fibroblast Growth Factor Signaling in Metabolic Regulation.

    PubMed

    Nies, Vera J M; Sancar, Gencer; Liu, Weilin; van Zutphen, Tim; Struik, Dicky; Yu, Ruth T; Atkins, Annette R; Evans, Ronald M; Jonker, Johan W; Downes, Michael Robert

    2015-01-01

    The prevalence of obesity is a growing health problem. Obesity is strongly associated with several comorbidities, such as non-alcoholic fatty liver disease, certain cancers, insulin resistance, and type 2 diabetes, which all reduce life expectancy and life quality. Several drugs have been put forward in order to treat these diseases, but many of them have detrimental side effects. The unexpected role of the family of fibroblast growth factors in the regulation of energy metabolism provides new approaches to the treatment of metabolic diseases and offers a valuable tool to gain more insight into metabolic regulation. The known beneficial effects of FGF19 and FGF21 on metabolism, together with recently discovered similar effects of FGF1 suggest that FGFs and their derivatives carry great potential as novel therapeutics to treat metabolic conditions. To facilitate the development of new therapies with improved targeting and minimal side effects, a better understanding of the molecular mechanism of action of FGFs is needed. In this review, we will discuss what is currently known about the physiological roles of FGF signaling in tissues important for metabolic homeostasis. In addition, we will discuss current concepts regarding their pharmacological properties and effector tissues in the context of metabolic disease. Also, the recent progress in the development of FGF variants will be reviewed. Our goal is to provide a comprehensive overview of the current concepts and consensuses regarding FGF signaling in metabolic health and disease and to provide starting points for the development of FGF-based therapies against metabolic conditions.

  17. [Growth factors in human tooth development].

    PubMed

    Bellone, C; Barni, T; Pagni, L; Balboni, G C; Vannelli, G B

    1990-03-01

    Our research concerns the immunohistochemical localization of EGF and IGF-I receptors in the tooth germ, using monoclonal antibodies. The results show that in the early phases of human tooth development EGF and IGF-I receptors are present. At bud stage both receptors are localized at dental laminae level, in some epithelial cells of the tooth bud and in some mesenchymal cells. At cap stage the receptors are present in the outer and inner enamel epithelium, and in some cells of stellate reticulum. As far as concerns the mesenchymal cells, some cells of dental papilla in contact with enamel organ, are intensely positive. The immunopositivity is present also in some mesenchymal cells at follicular level. At late cap stage and at early bell stage receptors are not present at inner enamel epithelium level but they can be detectable in the mesenchyma of dental papilla and in some cells of the follicle. On the basis of these results it may be hypothesized that EGF and IGF-I can act as growth factors in the modulation of cellular proliferation and differentiation during the human tooth morphogenesis. Moreover, it is possible that these substances can play a role in the mesenchymal-epithelial interaction in the developing human tooth.

  18. Transforming growth factor beta1 and aldosterone

    PubMed Central

    Matsuki, Kota; Hathaway, Catherine K.; Chang, Albert S.; Smithies, Oliver; Kakoki, Masao

    2016-01-01

    Purpose of review It is well established that blocking renin-angiotensin II-aldosterone system (RAAS) is effective for the treatment of cardiovascular and renal complications in hypertension and diabetes mellitus. Although the induction of transforming growth factor beta1 (TGFbeta1) by components of RAAS mediates the hypertrophic and fibrogenic changes in cardiovascular-renal complications, it is still controversial as to whether TGFbeta1 can be a target to prevent such complications. Here we review recent findings on the role of TGFbeta1 in fluid homeostasis, focusing on the relationship with aldosterone. Recent findings TGFbeta1 suppresses adrenal production of aldosterone and renal tubular sodium reabsorption. We have generated mice with TGFbeta1 mRNA expression graded in five steps from 10% to 300% normal, and found that blood pressure and plasma volume are negatively regulated by TGFbeta1. Notably, the 10 % hypomorph exhibits primary aldosteronism and sodium and water retention due to markedly impaired urinary excretion of water and electrolytes. Summary These results identify TGFbeta signaling as an important counterregulatory system against aldosterone. Understanding the molecular mechanisms for the suppressive effects of TGFbeta1 on adrenocortical and renal function may further our understanding of primary aldosteronism as well as assist in the development of novel therapeutic strategies for hypertension. PMID:25587902

  19. Induction of differentiation in psoriatic keratinocytes by propylthiouracil and fructose.

    PubMed

    Arul, Santhosh; Dayalan, Haripriya; Jegadeesan, Muhilan; Damodharan, Prabhavathy

    2016-12-01

    Psoriasis is characterized by uncontrolled proliferation and poor differentiation. Sirtuin1 (SIRT1) a class III deacetylase, crucial for differentiation in normal keratinocytes, is reduced in psoriasis. Down regulated SIRT1 levels may contribute to poor differentiation in psoriasis. In addition, the levels of early differentiation factors Keratin1 (K1) and Keratin10 (K10) are depleted in psoriasis. We attempted to study a possible effect of fructose, a SIRT1 upregulator and Propylthiouracil (PTU) to augment differentiation in psoriatic keratinocytes. Keratinocytes were cultured from lesional biopsies obtained from psoriatic patients and control cells were obtained from patients undergoing abdominoplasty. Cells were treated with fructose and PTU individually. K1 and K10 transcript levels were measured to evaluate early differentiation; SIRT1 protein expression was also studied to decipher its role in the mechanism of differentiation. The K1, K10 transcript levels, SIRT1 protein and transcript levels in fructose treated psoriatic keratinocytes were improved. This suggests keratinocyte differentiation was induced by fructose through SIRT1 upregulation. Whereas PTU induced differentiation, as confirmed by improved K1, K10 transcript levels followed a non-SIRT1 mechanism. We conclude that the use of fructose and PTU may be an adjunct to the existing therapies for psoriasis. PMID:27453822

  20. Fibroblast Growth Factor Homologous Factors Modulate Cardiac Calcium Channels

    PubMed Central

    Hennessey, Jessica A.; Wei, Eric Q.; Pitt, Geoffrey S.

    2013-01-01

    Rationale Fibroblast growth factor (FGF) homologous factors (FHFs, FGF11-14) are intracellular modulators of voltage-gated Na+ channels, but their cellular distribution in cardiomyocytes indicated that they performed other functions. Objective We aimed to uncover novel roles for FHFs in cardiomyocytes starting with a proteomic approach to identify novel interacting proteins. Methods and Results Affinity purification of FGF13 from rodent ventricular lysates followed by mass spectroscopy revealed an interaction with Junctophilin-2, a protein that organizes the close apposition of the L-type Ca2+ channel, CaV1.2, and the ryanodine receptor, RyR2, in the dyad. Immunocytochemical analysis revealed overall T-tubule structure and localization RyR2 were unaffected by FGF13 knockdown in adult ventricular cardiomyocytes, but localization of CaV1.2 was affected. FGF13 knockdown decreased CaV1.2 current density, and reduced the amount of CaV1.2 at the surface due to aberrant localization of the channels. CaV1.2 current density and channel localization were rescued by expression of an shRNA-insensitive FGF13, indicating a specific role for FGF13. Consistent with these newly discovered effects on CaV1.2, we demonstrated that FGF13 also regulated Ca2+-induced Ca2+ release, indicated by a smaller Ca2+ transient after FGF13 knockdown. Further, FGF13 knockdown caused a profound decrease in the cardiac action potential half width. Conclusions This study demonstrates that FHFs are not only potent modulators voltage-gated Na+ channels, but also affect Ca2+ channels and their function. We predict that FHF loss-of-function mutations would adversely affect currents through both Na+ and Ca2+ channels, suggesting that FHFs may be arrhythmogenic loci, leading to arrhythmias through a novel, dual-ion channel mechanism. PMID:23804213

  1. Actin filament dynamics impacts keratinocyte stem cell maintenance

    PubMed Central

    Nanba, Daisuke; Toki, Fujio; Matsushita, Natsuki; Matsushita, Sachi; Higashiyama, Shigeki; Barrandon, Yann

    2013-01-01

    Cultured human epidermal keratinocyte stem cells (holoclones) are crucial for regenerative medicine for burns and genetic disorders. In serial culture, holoclones progressively lose their proliferative capacity to become transient amplifying cells with limited growth (paraclones), a phenomenon termed clonal conversion. Although it negatively impacts the culture lifespan and the success of cell transplantation, little is known on the molecular mechanism underlying clonal conversion. Here, we show that holoclones and paraclones differ in their actin filament organization, with actin bundles distributed radially in holoclones and circumferentially in paraclones. Moreover, actin organization sets the stage for a differing response to epidermal growth factor (EGF), since EGF signalling induces a rapid expansion of colony size in holoclones and a significant reduction in paraclones. Furthermore, inhibition of PI3K or Rac1 in holoclones results in the reorganization of actin filaments in a pattern that is similar to that of paraclones. Importantly, continuous Rac1 inhibition in holoclones results in clonal conversion and reduction of growth potential. Together, our data connect loss of stem cells to EGF-induced colony dynamics governed by Rac1. PMID:23554171

  2. Vascular endothelial growth factor in central nervous system injuries - a vascular growth factor getting nervous?

    PubMed

    Sköld, Mattias K; Kanje, Martin

    2008-11-01

    Vascular Endothelial Growth Factor (VEGF) is recognized as a central factor in growth, survival and permeability of blood vessels in both physiological and pathological conditions. It is as such of importance for vascular responses in various central nervous system (CNS) disorders. Accumulating evidence suggest that VEGF may also act as a neuroprotective and neurotrophic factor supporting neuronal survival and neuronal regeneration. Findings of neuropilins as shared co-receptors between molecules with such seemingly different functions as the axon guidance molecules semaphorins and VEGF has further boosted the interest in the role of VEGF in neural tissue injury and repair mechanisms. Thus, VEGF most likely act in parallel or concurrent on cells in both the vascular and nervous system. The present review gives a summary of known or potential aspects of the VEGF system in the healthy and diseased nervous system. The potential benefits but also problems and pitfalls in intervening in the actions of such a multifunctional factor as VEGF in the disordered CNS are also covered.

  3. Dynamic Transcriptional and Epigenetic Regulation of Human Epidermal Keratinocyte Differentiation

    PubMed Central

    Cavazza, Alessia; Miccio, Annarita; Romano, Oriana; Petiti, Luca; Malagoli Tagliazucchi, Guidantonio; Peano, Clelia; Severgnini, Marco; Rizzi, Ermanno; De Bellis, Gianluca; Bicciato, Silvio; Mavilio, Fulvio

    2016-01-01

    Summary Human skin is maintained by the differentiation and maturation of interfollicular stem and progenitors cells. We used DeepCAGE, genome-wide profiling of histone modifications and retroviral integration analysis, to map transcripts, promoters, enhancers, and super-enhancers (SEs) in prospectively isolated keratinocytes and transit-amplifying progenitors, and retrospectively defined keratinocyte stem cells. We show that >95% of the active promoters are in common and differentially regulated in progenitors and differentiated keratinocytes, while approximately half of the enhancers and SEs are stage specific and account for most of the epigenetic changes occurring during differentiation. Transcription factor (TF) motif identification and correlation with TF binding site maps allowed the identification of TF circuitries acting on enhancers and SEs during differentiation. Overall, our study provides a broad, genome-wide description of chromatin dynamics and differential enhancer and promoter usage during epithelial differentiation, and describes a novel approach to identify active regulatory elements in rare stem cell populations. PMID:27050947

  4. DNA repair in cultured keratinocytes.

    PubMed

    Liu, S C; Parsons, S; Hanawalt, P C

    1983-07-01

    Most of our understanding of DNA repair mechanisms in human cells has come from the study of these processes in cultured fibroblasts. The unique properties of keratinocytes and their pattern of terminal differentiation led us to a comparative examination of their DNA repair properties. We have examined the relative repair capabilities of the basal cells and the differentiated epidermal keratinocytes as well as possible correlations of DNA repair capacity with respect to age of the donor. In addition, since portions of human skin are chronically exposed to sunlight, we have assessed the repair response to ultraviolet (UV) irradiation (254 nm) when the cells are conditioned by chronic low-level UV irradiation. The methods of Liu and Karasek were used to grow pure keratinocytes on collagen gels following their isolation from abdominal skin of newborns and adults at autopsy. Density labeling with 5-bromodeoxyuridine was used to resolve repair replication from the semiconservative mode. We found similar repair characteristics in human epidermal keratinocytes to those previously reported for cultured fibroblasts. However, the DNA repair response in basal cells was much greater than that in differentiated cells from the same skin preparation. Our comparative studies of DNA repair in keratinocytes from infant and aged donors have revealed no significant age-related differences for repair of UV-induced damage to DNA. Sublethal UV conditioning of cells from infant skin had no appreciable effect on either the repair or normal replication response to higher, challenge doses of UVL. However, such conditioning resulted in attenuated repair in keratinocytes from adult skin after UV doses above 25 J/m2. In addition, a surprising enhancement in replication was seen in conditioned cells from adult following challenge UV doses.

  5. Oncogenic Human Papillomaviruses Activate the Tumor-Associated Lens Epithelial-Derived Growth Factor (LEDGF) Gene

    PubMed Central

    Leitz, Jenny; Reuschenbach, Miriam; Lohrey, Claudia; Honegger, Anja; Accardi, Rosita; Tommasino, Massimo; Llano, Manuel; von Knebel Doeberitz, Magnus; Hoppe-Seyler, Karin; Hoppe-Seyler, Felix

    2014-01-01

    The expression of the human papillomavirus (HPV) E6/E7 oncogenes is crucial for HPV-induced malignant cell transformation. The identification of cellular targets attacked by the HPV oncogenes is critical for our understanding of the molecular mechanisms of HPV-associated carcinogenesis and may open novel therapeutic opportunities. Here, we identify the Lens Epithelial-Derived Growth Factor (LEDGF) gene as a novel cellular target gene for the HPV oncogenes. Elevated LEDGF expression has been recently linked to human carcinogenesis and can protect tumor cells towards different forms of cellular stress. We show that intracellular LEDGF mRNA and protein levels in HPV-positive cancer cells are critically dependent on the maintenance of viral oncogene expression. Ectopic E6/E7 expression stimulates LEDGF transcription in primary keratinocytes, at least in part via activation of the LEDGF promoter. Repression of endogenous LEDGF expression by RNA interference results in an increased sensitivity of HPV-positive cancer cells towards genotoxic agents. Immunohistochemical analyses of cervical tissue specimens reveal a highly significant increase of LEDGF protein levels in HPV-positive lesions compared to histologically normal cervical epithelium. Taken together, these results indicate that the E6/E7-dependent maintenance of intracellular LEDGF expression is critical for protecting HPV-positive cancer cells against various forms of cellular stress, including DNA damage. This could support tumor cell survival and contribute to the therapeutic resistance of cervical cancers towards genotoxic treatment strategies in the clinic. PMID:24604027

  6. Genetic programs of epithelial cell plasticity directed by transforming growth factor

    PubMed Central

    Zavadil, Jiri; Bitzer, Markus; Liang, Dan; Yang, Yaw-Ching; Massimi, Aldo; Kneitz, Susanne; Piek, Ester; Böttinger, Erwin P.

    2001-01-01

    Epithelial–mesenchymal transitions (EMTs) are an essential manifestation of epithelial cell plasticity during morphogenesis, wound healing, and tumor progression. Transforming growth factor-β (TGF-β) modulates epithelial plasticity in these physiological contexts by inducing EMT. Here we report a transcriptome screen of genetic programs of TGF-β-induced EMT in human keratinocytes and propose functional roles for extracellular response kinase (ERK) mitogen-activated protein kinase signaling in cell motility and disruption of adherens junctions. We used DNA arrays of 16,580 human cDNAs to identify 728 known genes regulated by TGF-β within 4 hours after treatment. TGF-β-stimulated ERK signaling mediated regulation of 80 target genes not previously associated with this pathway. This subset is enriched for genes with defined roles in cell–matrix interactions, cell motility, and endocytosis. ERK-independent genetic programs underlying the onset of EMT involve key pathways and regulators of epithelial dedifferentiation, undifferentiated transitional and mesenchymal progenitor phenotypes, and mediators of cytoskeletal reorganization. The gene expression profiling approach delineates complex context-dependent signaling pathways and transcriptional events that determine epithelial cell plasticity controlled by TGF-β. Investigation of the identified pathways and genes will advance the understanding of molecular mechanisms that underlie tumor invasiveness and metastasis. PMID:11390996

  7. Basic fibroblast growth factor promotes melanocyte migration via increased expression of p125(FAK) on melanocytes.

    PubMed

    Wu, Ching-Shuang; Lan, Cheng-Che E; Chiou, Min-Hsi; Yu, Hsin-Su

    2006-01-01

    Vitiligo is an acquired pigmentary disorder characterized by depigmentation of skin and hair. Melanocyte migration is an important event in re-pigmentation of vitiligo. We have demonstrated that narrow-band ultraviolet B (UVB) irradiation stimulated cultured keratinocytes to release a significant amount of basic fibroblast growth factor (bFGF). Furthermore, narrow-band UVB enhanced migration of melanocytes via increased expression of phosphorylated focal adhesion kinase (p125(FAK)) on melanocytes. The aim of this study was to investigate the effect of recombinant human bFGF (rhbFGF) on melanocyte migration. The relationship between the expression of p125(FAK) and melanocyte migration induced by rhbFGF was also studied. Our results demonstrated that rhbFGF significantly enhanced migration of melanocytes and p125(FAK) expression on melanocytes. Herbimycin A, a potent p125(FAK) inhibitor, effectively abolished rhbFGF-induced melanocyte migration. The combined results indicated that p125(FAK) plays an important role in the signal transduction pathway of melanocyte migration induced by bFGF.

  8. Oncogenic human papillomaviruses activate the tumor-associated lens epithelial-derived growth factor (LEDGF) gene.

    PubMed

    Leitz, Jenny; Reuschenbach, Miriam; Lohrey, Claudia; Honegger, Anja; Accardi, Rosita; Tommasino, Massimo; Llano, Manuel; von Knebel Doeberitz, Magnus; Hoppe-Seyler, Karin; Hoppe-Seyler, Felix

    2014-03-01

    The expression of the human papillomavirus (HPV) E6/E7 oncogenes is crucial for HPV-induced malignant cell transformation. The identification of cellular targets attacked by the HPV oncogenes is critical for our understanding of the molecular mechanisms of HPV-associated carcinogenesis and may open novel therapeutic opportunities. Here, we identify the Lens Epithelial-Derived Growth Factor (LEDGF) gene as a novel cellular target gene for the HPV oncogenes. Elevated LEDGF expression has been recently linked to human carcinogenesis and can protect tumor cells towards different forms of cellular stress. We show that intracellular LEDGF mRNA and protein levels in HPV-positive cancer cells are critically dependent on the maintenance of viral oncogene expression. Ectopic E6/E7 expression stimulates LEDGF transcription in primary keratinocytes, at least in part via activation of the LEDGF promoter. Repression of endogenous LEDGF expression by RNA interference results in an increased sensitivity of HPV-positive cancer cells towards genotoxic agents. Immunohistochemical analyses of cervical tissue specimens reveal a highly significant increase of LEDGF protein levels in HPV-positive lesions compared to histologically normal cervical epithelium. Taken together, these results indicate that the E6/E7-dependent maintenance of intracellular LEDGF expression is critical for protecting HPV-positive cancer cells against various forms of cellular stress, including DNA damage. This could support tumor cell survival and contribute to the therapeutic resistance of cervical cancers towards genotoxic treatment strategies in the clinic.

  9. Fibroblast Growth Factor Signaling in Metabolic Regulation

    PubMed Central

    Nies, Vera J. M.; Sancar, Gencer; Liu, Weilin; van Zutphen, Tim; Struik, Dicky; Yu, Ruth T.; Atkins, Annette R.; Evans, Ronald M.; Jonker, Johan W.; Downes, Michael Robert

    2016-01-01

    The prevalence of obesity is a growing health problem. Obesity is strongly associated with several comorbidities, such as non-alcoholic fatty liver disease, certain cancers, insulin resistance, and type 2 diabetes, which all reduce life expectancy and life quality. Several drugs have been put forward in order to treat these diseases, but many of them have detrimental side effects. The unexpected role of the family of fibroblast growth factors in the regulation of energy metabolism provides new approaches to the treatment of metabolic diseases and offers a valuable tool to gain more insight into metabolic regulation. The known beneficial effects of FGF19 and FGF21 on metabolism, together with recently discovered similar effects of FGF1 suggest that FGFs and their derivatives carry great potential as novel therapeutics to treat metabolic conditions. To facilitate the development of new therapies with improved targeting and minimal side effects, a better understanding of the molecular mechanism of action of FGFs is needed. In this review, we will discuss what is currently known about the physiological roles of FGF signaling in tissues important for metabolic homeostasis. In addition, we will discuss current concepts regarding their pharmacological properties and effector tissues in the context of metabolic disease. Also, the recent progress in the development of FGF variants will be reviewed. Our goal is to provide a comprehensive overview of the current concepts and consensuses regarding FGF signaling in metabolic health and disease and to provide starting points for the development of FGF-based therapies against metabolic conditions. PMID:26834701

  10. Novel Drosophila receptor that binds multiple growth factors

    SciTech Connect

    Rosner, M.R.; Thompson, K.L.; Garcia, V.; Decker, S.J.

    1986-05-01

    The authors have recently reported the identification of a novel growth factor receptor from Drosophila cell cultures that has dual binding specificity for both insulin and epidermal growth factor (EGF). This 100 kDa protein is also antigenically related to the cytoplasmic region of the mammalian EGF receptor-tyrosine kinase. They now report that this protein binds to mammalian nerve growth factor and human transforming growth factor alpha as well as insulin and EGF with apparent dissociation constants ranging from 10/sup -6/ to 10/sup -8/ M. The 100 kDa protein can be affinity-labeled with these /sup 125/I-labeled growth factors after immunoprecipitation with anti-EGF receptor antiserum. These four growth factors appear to share a common binding site, as evidenced by their ability to block affinity labelling by /sup 125/I-insulin. No significant binding to the 100 kDa protein was observed with platelet-derived growth factor, transforming growth factor beta, or glucagon. The 100 kDa Drosophila protein has a unique ligand-binding spectrum with no direct counterpart in mammalian cells and may represent an evolutionary precursor of the mammalian receptors for these growth factors.

  11. Growth factor array fabrication using a color ink jet printer.

    PubMed

    Watanabe, Kohei; Miyazaki, Takeshi; Matsuda, Ryoichi

    2003-04-01

    We have developed a novel method for growth factor analysis using a commercial color ink jet printer to fabricate substrata patterned with growth factors. We prepared substrata with insulin printed in a simple pattern or containing multiple areas of varying quantities of printed insulin. When we cultured the mouse myoblast cell line, C2C12, on the insulin-patterned substrata, the cells were grown in the same pattern with the insulin-printed pattern. Cell culture with the latter substrata demonstrated that quantity control of insulin deposition by a color ink jet printer is possible. For further applications, we developed substrata with insulin-like growth factor-I (IGF-I) and basic fibroblast growth factor (bFGF) spotted in 16 different areas in varying combinations and concentrations (growth factor array). With this growth factor array, C2C12 cells were cultured, and the onset of muscle cell differentiation was monitored for the expression of the myogenic regulator myogenin. The ratio of cells expressing myogenin varied with the doses of IGF-I and bFGF in the sections, demonstrating a feasibility of growth factor array fabrication by a color ink jet printer. Since a printer manipulates several colors, this method can be easily applied to multivariate analyses of growth factors and attachment factors affecting cell growth and differentiation. This method may provide a powerful tool for cell biology and tissue engineering, especially for stem cell research in investigating unknown conditions for differentiation.

  12. Identification of the ETA receptor subtype that mediates endothelin induced autocrine proliferation of normal human keratinocytes.

    PubMed

    Bagnato, A; Venuti, A; Di Castro, V; Marcante, M L

    1995-04-01

    Endothelin-1 has a wide range of pharmacological effects in various tissues and acts as autocrine/paracrine factor. The potential of ET-1 to function as an autocrine growth factor was evaluated in normal human keratinocytes. Radioligand binding studies showed that 125I-ET-1 bound to a single class of high-affinity-binding sites on the surface of the cells. The dissociation constant was 0.045 nM with receptor numbers of 1700 sites/cell. Treatment with serum caused increases in expression of binding sites (3500 sites/cell), with no change in binding affinity. ET-1 stimulated thymidine incorporation in these cells that expressed ET receptors. An ET antagonist selective for the ETA receptor subtype (BQ 123) inhibited DNA synthesis stimulated by ET-1 and reduced the basal growth rate of unstimulated cells. These data suggest that the ET-1 induced DNA synthesis is mediated by ETA receptor subtype and that endogenously produced ET-1 promotes the autocrine proliferation of keratinocytes.

  13. Absence of premature senescence in Werner's syndrome keratinocytes.

    PubMed

    Ibrahim, Badr; Sheerin, Angela N; Jennert-Burston, Katrin; Bird, Joe L E; Massala, M V; Illsley, Matthew; James, S Elizabeth; Faragher, Richard G A

    2016-10-01

    Werner's syndrome (WS) is an autosomal recessive genetic disorder caused by loss of function mutation in wrn and is a useful model of premature in vivo ageing. Cellular senescence is a plausible causal mechanism of mammalian ageing and, at the cellular level, WS fibroblasts show premature senescence resulting from a combination of telomeric attrition and replication fork stalling. Over 90% of WS fibroblast cultures achieve <20 population doublings (PD) in vitro compared to wild type human fibroblast cultures. It has been proposed that some cell types, capable of proliferation, will fail to show a premature senescence phenotype in response to wrn mutations. To test this hypothesis, human dermal keratinocytes (derived from both WS and wild type patients) were cultured long term. WS Keratinocytes showed a replicative lifespan in excess of 100 population doublings but maintained functional growth arrest mechanisms based on p16 and p53. The karyotype of the cells was superficially normal and the cultures retained markers characteristic of keratinocyte holoclones (stem cells) including p63 expression and telomerase activity. Accordingly we conclude that, in contrast to WS fibroblasts, WS keratinocytes do not demonstrate slow growth rates or features of premature senescence. These findings suggest that the epidermis is among the tissue types that do not display symptoms of premature ageing caused by loss of function of wrn. This is in support that Werner's syndrome is a segmental progeroid syndrome. PMID:27492502

  14. Induction of Hair Growth by Insulin-Like Growth Factor-1 in 1,763 MHz Radiofrequency-Irradiated Hair Follicle Cells

    PubMed Central

    Jo, Seong Jin; Cho, A-Ri; Jeon, Soon-Ik; Choi, Hyung-Do; Kim, Kyu Han; Park, Gun-Sik; Pack, Jeong-Ki; Kwon, Oh Sang; Park, Woong-Yang

    2011-01-01

    Radiofrequency (RF) radiation does not transfer high energy to break the covalent bonds of macromolecules, but these low energy stimuli might be sufficient to induce molecular responses in a specific manner. We monitored the effect of 1,763 MHz RF radiation on cultured human dermal papilla cells (hDPCs) by evaluating changes in the expression of cytokines related to hair growth. The expression of insulin-like growth factor-1 (IGF-1) mRNA in hDPCs was significantly induced upon RF radiation at the specific absorption rate of 10 W/kg, which resulted in increased expression of B-cell chronic lymphocytic leukemia/lymphoma 2 (BCL-2) and cyclin D1 (CCND1) proteins and increased phosphorylation of MAPK1 protein. Exposure to 10 W/kg RF radiation 1 h per day for 7 days significantly enhanced hair shaft elongation in ex vivo hair organ cultures. In RF-exposed follicular matrix keratinocytes in the hair bulb, the expression of Ki-67 was increased, while the signal for terminal deoxynucleotidyl transferase dUTP nick end labeling was reduced. From these results, we suggest that 1,763 MHz RF exposure stimulates hair growth in vitro through the induction of IGF-1 in hDPCs. PMID:22164296

  15. Directed random walks and constraint programming reveal active pathways in hepatocyte growth factor signaling.

    PubMed

    Kittas, Aristotelis; Delobelle, Aurélien; Schmitt, Sabrina; Breuhahn, Kai; Guziolowski, Carito; Grabe, Niels

    2016-01-01

    An effective means to analyze mRNA expression data is to take advantage of established knowledge from pathway databases, using methods such as pathway-enrichment analyses. However, pathway databases are not case-specific and expression data could be used to infer gene-regulation patterns in the context of specific pathways. In addition, canonical pathways may not always describe the signaling mechanisms properly, because interactions can frequently occur between genes in different pathways. Relatively few methods have been proposed to date for generating and analyzing such networks, preserving the causality between gene interactions and reasoning over the qualitative logic of regulatory effects. We present an algorithm (MCWalk) integrated with a logic programming approach, to discover subgraphs in large-scale signaling networks by random walks in a fully automated pipeline. As an exemplary application, we uncover the signal transduction mechanisms in a gene interaction network describing hepatocyte growth factor-stimulated cell migration and proliferation from gene-expression measured with microarray and RT-qPCR using in-house perturbation experiments in a keratinocyte-fibroblast co-culture. The resulting subgraphs illustrate possible associations of hepatocyte growth factor receptor c-Met nodes, differentially expressed genes and cellular states. Using perturbation experiments and Answer Set programming, we are able to select those which are more consistent with the experimental data. We discover key regulator nodes by measuring the frequency with which they are traversed when connecting signaling between receptors and significantly regulated genes and predict their expression-shift consistently with the measured data. The Java implementation of MCWalk is publicly available under the MIT license at: https://bitbucket.org/akittas/biosubg.

  16. Presence of growth factors in palmar and plantar fibromatoses.

    PubMed

    Zamora, R L; Heights, R; Kraemer, B A; Erlich, H P; Groner, J P

    1994-05-01

    Palmar and plantar fibromatoses are disease processes in which the presence of certain growth factors has not been defined. Monoclonal antibodies against transforming growth factor-beta, epidermal growth factor, procollagen type 1, fibronectin, phosphotyrosine residues, and CD41 platelet antigen were used in standard immunoperoxidase staining to study 36 nodules and 24 cords obtained from patients with fibromatoses. The specimens were studied via light microscopy, and staining intensity was quantitated using a computer-enhanced video system. Transforming growth factor-beta staining paralleled procollagen I, fibronectin, and phosphotyrosine staining within the nodule (early stages) but not the cord (late stages) tissue. These factors showed significant increased staining in the early stage of fibromatosis when compared to the late stage. This study is a preliminary demonstration of the presence of transforming growth factor-beta in palmar and plantar fibromatoses.

  17. Clinical application of growth factors and cytokines in wound healing.

    PubMed

    Barrientos, Stephan; Brem, Harold; Stojadinovic, Olivera; Tomic-Canic, Marjana

    2014-01-01

    Wound healing is a complex and dynamic biological process that involves the coordinated efforts of multiple cell types and is executed and regulated by numerous growth factors and cytokines. There has been a drive in the past two decades to study the therapeutic effects of various growth factors in the clinical management of nonhealing wounds (e.g., pressure ulcers, chronic venous ulcers, diabetic foot ulcers). For this review, we conducted an online search of Medline/PubMed and critically analyzed the literature regarding the role of growth factors and cytokines in the management of these wounds. We focused on currently approved therapies, emerging therapies, and future research possibilities. In this review, we discuss four growth factors and cytokines currently being used on and off label for the healing of wounds. These include granulocyte-macrophage colony-stimulating factor, platelet-derived growth factor, vascular endothelial growth factor, and basic fibroblast growth factor. While the clinical results of using growth factors and cytokines are encouraging, many studies involved a small sample size and are disparate in measured endpoints. Therefore, further research is required to provide definitive evidence of efficacy.

  18. PCB153 reduces telomerase activity and telomere length in immortalized human skin keratinocytes (HaCaT) but not in human foreskin keratinocytes (NFK)

    SciTech Connect

    Senthilkumar, P.K.; Robertson, L.W.; Ludewig, G.

    2012-02-15

    Polychlorinated biphenyls (PCBs), ubiquitous environmental pollutants, are characterized by long term-persistence in the environment, bioaccumulation, and biomagnification in the food chain. Exposure to PCBs may cause various diseases, affecting many cellular processes. Deregulation of the telomerase and the telomere complex leads to several biological disorders. We investigated the hypothesis that PCB153 modulates telomerase activity, telomeres and reactive oxygen species resulting in the deregulation of cell growth. Exponentially growing immortal human skin keratinocytes (HaCaT) and normal human foreskin keratinocytes (NFK) were incubated with PCB153 for 48 and 24 days, respectively, and telomerase activity, telomere length, superoxide level, cell growth, and cell cycle distribution were determined. In HaCaT cells exposure to PCB153 significantly reduced telomerase activity, telomere length, cell growth and increased intracellular superoxide levels from day 6 to day 48, suggesting that superoxide may be one of the factors regulating telomerase activity, telomere length and cell growth compared to untreated control cells. Results with NFK cells showed no shortening of telomere length but reduced cell growth and increased superoxide levels in PCB153-treated cells compared to untreated controls. As expected, basal levels of telomerase activity were almost undetectable, which made a quantitative comparison of treated and control groups impossible. The significant down regulation of telomerase activity and reduction of telomere length by PCB153 in HaCaT cells suggest that any cell type with significant telomerase activity, like stem cells, may be at risk of premature telomere shortening with potential adverse health effects for the affected organism. -- Highlights: ► Human immortal (HaCaT) and primary (NFK) keratinocytes were exposed to PCB153. ► PCB153 significantly reduced telomerase activity and telomere length in HaCaT. ► No effect on telomere length and

  19. DNA repair in cultured keratinocytes

    SciTech Connect

    Liu, S.C.; Parsons, S.; Hanawalt, P.C.

    1983-07-01

    Most of our understanding of DNA repair mechanisms in human cells has come from the study of these processes in cultured fibroblasts. The unique properties of keratinocytes and their pattern of terminal differentiation led us to a comparative examination of their DNA repair properties. The relative repair capabilities of the basal cells and the differentiated epidermal keratinocytes as well as possible correlations of DNA repair capacity with respect to age of the donor have been examined. In addition, since portions of human skin are chronically exposed to sunlight, the repair response to ultraviolet (UV) irradiation (254 nm) when the cells are conditioned by chronic low-level UV irradiation has been assessed. The comparative studies of DNA repair in keratinocytes from infant and aged donors have revealed no significant age-related differences for repair of UV-induced damage to DNA. Sublethal UV conditioning of cells from infant skin had no appreciable effect on either the repair or normal replication response to higher, challenge doses of UVL. However, such conditioning resulted in attenuated repair in keratinocytes from adult skin after UV doses above 25 J/m2. In addition, a surprising enhancement in replication was seen in conditioned cells from adult following challenge UV doses.

  20. Growth factor effects on costal chondrocytes for tissue engineering fibrocartilage.

    PubMed

    Johns, D E; Athanasiou, K A

    2008-09-01

    Tissue-engineered fibrocartilage could become a feasible option for replacing tissues such as the knee meniscus or temporomandibular joint disc. This study employed five growth factors (insulin-like growth factor-I, transforming growth factor-beta1, epidermal growth factor, platelet-derived growth factor-BB, and basic fibroblast growth factor) in a scaffoldless approach with costal chondrocytes, attempting to improve biochemical and mechanical properties of engineered constructs. Samples were quantitatively assessed for total collagen, glycosaminoglycans, collagen type I, collagen type II, cells, compressive properties, and tensile properties at two time points. Most treated constructs had lower biomechanical and biochemical properties than the controls with no growth factors, suggesting a detrimental effect, but the treatment with insulin-like growth factor-I tended to improve the constructs. Additionally, the 6-week time point was consistently better than that at 3 weeks, with total collagen, glycosaminoglycans, and aggregate modulus doubling during this time. Further optimization of the time in culture and exogenous stimuli will be important in making a more functional replacement tissue. PMID:18597118

  1. Delivery of growth factors for tissue regeneration and wound healing.

    PubMed

    Koria, Piyush

    2012-06-01

    Growth factors are soluble secreted proteins capable of affecting a variety of cellular processes important for tissue regeneration. Consequently, the self-healing capacity of patients can be augmented by artificially enhancing one or more processes important for healing through the application of growth factors. However, their application in clinics remains limited due to lack of robust delivery systems and biomaterial carriers. Interestingly, all clinically approved therapies involving growth factors utilize some sort of a biomaterial carrier for growth factor delivery. This suggests that biomaterial delivery systems are extremely important for successful usage of growth factors in regenerative medicine. This review outlines the role of growth factors in tissue regeneration, and their application in both pre-clinical animal models of regeneration and clinical trials is discussed. Additionally, current status of biomaterial substrates and sophisticated delivery systems such as nanoparticles for delivery of exogenous growth factors and peptides in humans are reviewed. Finally, issues and possible future research directions for growth factor therapy in regenerative medicine are discussed.

  2. Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth

    PubMed Central

    1992-01-01

    Hepatocyte Growth Factor (HGF, also known as Scatter Factor) is a powerful mitogen or motility factor in different cells, acting through the tyrosine kinase receptor encoded by the MET protooncogene. Endothelial cells express the MET gene and expose at the cell surface the mature protein (p190MET) made of a 50 kD (alpha) subunit disulfide linked to a 145-kD (beta) subunit. HGF binding to endothelial cells identifies two sites with different affinities. The higher affinity binding site (Kd = 0.35 nM) corresponds to the p190MET receptor. Sub- nanomolar concentrations of HGF, but not of a recombinant inactive precursor, stimulate the receptor kinase activity, cell proliferation and motility. HGF induces repairs of a wound in endothelial cell monolayer. HGF stimulates the scatter of endothelial cells grown on three-dimensional collagen gels, inducing an elongated phenotype. In the rabbit cornea, highly purified HGF promotes neovascularization at sub-nanomolar concentrations. HGF lacks activities related to hemostasis-thrombosis, inflammation and endothelial cells accessory functions. These data show that HGF is an in vivo potent angiogenic factor and in vitro induces endothelial cells to proliferate and migrate. PMID:1383237

  3. Expression of vascular endothelial growth factor and basic fibroblast growth factor in extramammary Paget disease

    PubMed Central

    Xu, Xiaoyun; Shao, Ning; Qiao, Di; Wang, Zengjun; Song, Ningjing; Song, Ninghong

    2015-01-01

    Extramammary Paget’s disease (EMPD) is a special type of cancers. The etiology of the disease is still unclear. We aimed to study the expression differences of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in EMPD tissues and corresponding adjacent normal tissues. The mRNA expression was detected by RT-PCR and the protein expression was explored by immunohistochemistry. Higher immunostaining signal scores of bFGF and VEGF in EMPD tissues had been found (z = -3.827, P < 0.001, z = -3.729, P < 0.001, respectively). In addition, the mRNA expression of bFGF and VEGF was higher in EMPD tissues, which had been validated by RT-PCR (t = 5.771, P < 0.001, t = 3.304, P = 0.004, respectively). The VEGF and bFGF might be the key signaling proteins in angiogenesis of EMPD. How to block the VEGF and bFGF in EMPD and to destroy the blood supply of the tumor cells becomes the focus of our future research. PMID:26045818

  4. Non-thermal Plasma Activates Human Keratinocytes by Stimulation of Antioxidant and Phase II Pathways

    PubMed Central

    Schmidt, Anke; Dietrich, Stephan; Steuer, Anna; Weltmann, Klaus-Dieter; von Woedtke, Thomas; Masur, Kai; Wende, Kristian

    2015-01-01

    Non-thermal atmospheric pressure plasma provides a novel therapeutic opportunity to control redox-based processes, e.g. wound healing, cancer, and inflammatory diseases. By spatial and time-resolved delivery of reactive oxygen and nitrogen species, it allows stimulation or inhibition of cellular processes in biological systems. Our data show that both gene and protein expression is highly affected by non-thermal plasma. Nuclear factor erythroid-related factor 2 (NRF2) and phase II enzyme pathway components were found to act as key controllers orchestrating the cellular response in keratinocytes. Additionally, glutathione metabolism, which is a marker for NRF2-related signaling events, was affected. Among the most robustly increased genes and proteins, heme oxygenase 1, NADPH-quinone oxidoreductase 1, and growth factors were found. The roles of NRF2 targets, investigated by siRNA silencing, revealed that NRF2 acts as an important switch for sensing oxidative stress events. Moreover, the influence of non-thermal plasma on the NRF2 pathway prepares cells against exogenic noxae and increases their resilience against oxidative species. Via paracrine mechanisms, distant cells benefit from cell-cell communication. The finding that non-thermal plasma triggers hormesis-like processes in keratinocytes facilitates the understanding of plasma-tissue interaction and its clinical application. PMID:25589789

  5. Non-thermal plasma activates human keratinocytes by stimulation of antioxidant and phase II pathways.

    PubMed

    Schmidt, Anke; Dietrich, Stephan; Steuer, Anna; Weltmann, Klaus-Dieter; von Woedtke, Thomas; Masur, Kai; Wende, Kristian

    2015-03-13

    Non-thermal atmospheric pressure plasma provides a novel therapeutic opportunity to control redox-based processes, e.g. wound healing, cancer, and inflammatory diseases. By spatial and time-resolved delivery of reactive oxygen and nitrogen species, it allows stimulation or inhibition of cellular processes in biological systems. Our data show that both gene and protein expression is highly affected by non-thermal plasma. Nuclear factor erythroid-related factor 2 (NRF2) and phase II enzyme pathway components were found to act as key controllers orchestrating the cellular response in keratinocytes. Additionally, glutathione metabolism, which is a marker for NRF2-related signaling events, was affected. Among the most robustly increased genes and proteins, heme oxygenase 1, NADPH-quinone oxidoreductase 1, and growth factors were found. The roles of NRF2 targets, investigated by siRNA silencing, revealed that NRF2 acts as an important switch for sensing oxidative stress events. Moreover, the influence of non-thermal plasma on the NRF2 pathway prepares cells against exogenic noxae and increases their resilience against oxidative species. Via paracrine mechanisms, distant cells benefit from cell-cell communication. The finding that non-thermal plasma triggers hormesis-like processes in keratinocytes facilitates the understanding of plasma-tissue interaction and its clinical application.

  6. Vascular Endothelial Growth Factor is a Secreted Angiogenic Mitogen

    NASA Astrophysics Data System (ADS)

    Leung, David W.; Cachianes, George; Kuang, Wun-Jing; Goeddel, David V.; Ferrara, Napoleone

    1989-12-01

    Vascular endothelial growth factor (VEGF) was purified from media conditioned by bovine pituitary folliculostellate cells (FC). VEGF is a heparin-binding growth factor specific for vascular endothelial cells that is able to induce angiogenesis in vivo. Complementary DNA clones for bovine and human VEGF were isolated from cDNA libraries prepared from FC and HL60 leukemia cells, respectively. These cDNAs encode hydrophilic proteins with sequences related to those of the A and B chains of platelet-derived growth factor. DNA sequencing suggests the existence of several molecular species of VEGF. VEGFs are secreted proteins, in contrast to other endothelial cell mitogens such as acidic or basic fibroblast growth factors and platelet-derived endothelial cell growth factor. Human 293 cells transfected with an expression vector containing a bovine or human VEGF cDNA insert secrete an endothelial cell mitogen that behaves like native VEGF.

  7. Transgenic Studies with a Keratin Promoter-Driven Growth Hormone Transgene: Prospects for Gene Therapy

    NASA Astrophysics Data System (ADS)

    Wang, Xiaoming; Zinkel, Sandra; Polonsky, Kenneth; Fuchs, Elaine

    1997-01-01

    Keratinocytes are potentially appealing vehicles for the delivery of secreted gene products because they can be transferred to human skin by the relatively simple procedure of grafting. Adult human keratinocytes can be efficiently propagated in culture with sufficient proliferative capacity to produce enough epidermis to cover the body surface of an average adult. However, the feasibility of delivering secreted proteins through skin grafting rests upon (i) the strength of the promoter in keratinocytes and (ii) the efficiency of protein transport through the basement membrane of the stratified epithelium and into the bloodstream. In this paper, we use transgenic technology to demonstrate that the activity of the human keratin 14 promoter remains high in adult skin and that keratinocyte-derived human growth hormone (hGH) can be produced, secreted, and transported to the bloodstream of mice with efficiency that is sufficient to exceed by an order of magnitude the circulating hGH concentration in growing children. Transgenic skin grafts from these adults continue to produce and secrete hGH stably, at ≈ 1/10 physiological levels in the bloodstream of nontransgenic recipient mice. These studies underscore the utility of the keratin 14 promoter for expressing foreign transgenes in keratinocytes and demonstrate that keratinocytes can be used as effective vehicles for transporting factors to the bloodstream and for eliciting metabolic changes. These findings have important implications for considering the keratinocyte as a possible vehicle for gene therapy.

  8. Material factors influencing metallic whisker growth

    NASA Astrophysics Data System (ADS)

    Rodekohr, Chad L.

    Whiskering refers to the formation of slender, long, metallic filaments, much thinner than a human hair, that grow on a metallic thin film surface. They are readily observed for pure and alloyed zinc (Zn), silver (Ag), cadmium (Cd), indium (In), and tin (Sn) surfaces. The longest reported whisker length is 4.5 mm long but most high-aspect ratio whiskers range from 1-500 mum. The focus of this research is upon Sn whiskers. Sn whiskers pose serious reliability problems for the electronics industry and are known to be the source of failure in a wide range of electronic devices, such as nuclear power facilities, heart pacemakers, commercial satellites, aviation radar, telecommunication equipment, and desktop computers. The problem with whiskering has been recently exacerbated by the worldwide shift to lead (Pb) free electronics and the continuing reduction in electrical contact pitches. A thorough understanding of the growth mechanism of Sn whiskers is urgently needed. Currently, there is no universally accepted model that explains the broad range of observations on whiskering. The goals of this research are: (1) to develop a more detailed understanding of the physical mechanisms leading to the initiation and growth of Sn whiskers and (2) to outline reasonable mitigation strategies that could be followed to reduce or eliminate the problem of Sn whiskers. The major contributions of this work are: (1) A reliable method for growing Sn whiskers with predictable incubation times has been developed and tested. (2) A surface oxide is not necessary for whisker growth. (3) Intermetallic compounds (IMC) are not necessary for whisker growth. (4) Smoother, not rougher, substrate surfaces promote whisker growth. (5) Whiskers grow under both compressive and tensile thin film stress states. (6) Whisker growth increases with externally applied compression and tension forces. (7) Sn whiskers are composed of pure Sn except for the expected thin, native Sn oxide on their surface. (8) For

  9. TERATOGENIC RESPONSES ARE MODULATED IN MICE LACKING EXPRESSION OF EPIDERMAL GROWTH FACTOR (EGF) AND TRANSFORMING GROWTH FACTOR-ALPHA (TGF)

    EPA Science Inventory

    TITLE:
    TERATOGENIC RESPONSES ARE MODULATED IN MICE LACKING EXPRESSION OF EPIDERMAL GROWTH FACTOR (EGF) AND TRANSFORMING GROWTH FACTOR-ALPHA (TGF). AUTHORS (ALL): Abbott, Barbara D.1; Best, Deborah S.1; Narotsky, Michael G.1. SPONSOR NAME: None INSTITUTIONS (ALL): 1. Repro Tox ...

  10. The discovery of basic fibroblast growth factor/fibroblast growth factor-2 and its role in haematological malignancies.

    PubMed

    Ribatti, Domenico; Vacca, Angelo; Rusnati, Marco; Presta, Marco

    2007-01-01

    Basic fibroblast growth factor/fibroblast growth factor-2 is one of the best characterized of the pro-angiogenic cytokines. This review describes its history, as well as its role in tumor angiogenesis associated with haematological malignancies, as traced by the main contributions to the international medical literature.

  11. Time-dependent release of growth factors from implant surfaces treated with plasma rich in growth factors.

    PubMed

    Sánchez-Ilárduya, María Belén; Trouche, Elodie; Tejero, Ricardo; Orive, Gorka; Reviakine, Ilya; Anitua, Eduardo

    2013-05-01

    Plasma rich in growth factors (PRGFs) technology is an autologous platelet-rich plasma approach that provides a pool of growth factors and cytokines that have been shown to increase tissue regeneration and accelerate dental implant osseointegration. In this framework, the spatiotemporal release of growth factors and the establishment of a provisional fibrin matrix are likely to be key aspects governing the stimulation of the early phases of tissue regeneration around implants. We investigated the kinetics of growth factor release at implant surfaces functionalized either with PRGFs or platelet-poor plasma and correlated the results obtained with the morphology of the resulting interfaces. Our main finding is that activation and clot formation favors longer residence times of the growth factors at the interfaces studied, probably due to their retention in the adsorbed fibrin matrix. The concentration of the platelet-derived growth factors above the interfaces becomes negligible after 2-4 days and is significantly higher in the case of activated interfaces than in the case of nonactivated ones, whereas that of the plasmatic hepatocyte growth factor is independent of platelet concentration and activation, and remains significant for up to 9 days. Platelet-rich plasma preparations should be activated to permit growth factor release and thereby facilitate implant surface osseointegration.

  12. EDUCATION AS A FACTOR IN ECONOMIC GROWTH.

    ERIC Educational Resources Information Center

    MACKERTICH, ALEX

    THE VALUE OF AN EDUCATION IN THE ECONOMIC GROWTH OF AN UNDERDEVELOPED COUNTRY (INDIA) WAS INVESTIGATED USING THE CASE STUDY APPROACH. DATA WERE GATHERED AT BOTH THE CENTRAL GOVERNMENT AND VILLAGE LEVELS THROUGH INTERVIEWS WITH INDIAN GOVERNMENT OFFICIALS AND FROM OFFICIAL GOVERNMENT PUBLICATIONS CONCERNING THE NATION'S EDUCATIONAL EFFORTS, AS…

  13. Transforming growth factor-alpha-induced transcriptional activation of the vascular permeability factor (VPF/VEGF) gene requires AP-2-dependent DNA binding and transactivation.

    PubMed Central

    Gille, J; Swerlick, R A; Caughman, S W

    1997-01-01

    The endothelial cell-specific mitogen vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) represents a central regulator of cutaneous angiogenesis. Increased VPF/VEGF expression has recently been reported in psoriatic skin and healing wounds, both conditions in which transforming growth factor-alpha (TGF alpha) and its ligand, the epidermal growth factor receptor, are markedly up-regulated. Since TGF alpha strongly induces VPF/VEGF synthesis in keratinocytes, TGF alpha-mediated VPF/VEGF expression is likely to play a significant role in the initiation and maintenance of increased vascular hyperpermeability and hyperproliferation in skin biology. The objectives of the present studies were to determine the molecular mechanisms responsible for TGF alpha-induced transcriptional activation of the VPF/VEGF gene. We have identified a GC-rich TGF alpha-responsive region between -88 bp and -65 bp of the VPF/VEGF promoter that is necessary for constitutive and TGF alpha-inducible transcriptional activation. In electrophoretic mobility shift assays, this region binds Sp1-dependent protein complexes constitutively and an additional TGF alpha-inducible protein complex that is distinct from Sp1 protein. Both AP-2 and Egr-1 transcription factors were detected as components of the TGF alpha-inducible protein complex in supershift EMSA studies. In co-transfection studies, an AP-2 but not an Egr-1 expression vector activated VPF/VEGF transcription, thus indicating that AP-2 protein is functionally important in TGF alpha-induced VPF/VEGF gene expression. By clarifying regulatory mechanisms that are critical for angiogenic processes in the skin, these studies may form the basis for new therapeutic strategies to modulate VPF/VEGF expression in cutaneous inflammation and wound healing. PMID:9049304

  14. A high-affinity receptor for urokinase plasminogen activator on human keratinocytes: characterization and potential modulation during migration.

    PubMed Central

    McNeill, H; Jensen, P J

    1990-01-01

    Low passage cultures of normal human keratinocytes produce several components of the plasminogen activator/plasmin proteolytic cascade, including urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), and two specific inhibitors. Studies here presented demonstrate that these cells also contain a high-affinity (Kd = 3 x 10(-10) M) plasma membrane-binding site for uPA. High molecular weight uPA, either as the single-chain precursor or two-chain activated form, bound to the receptor; however, low molecular weight (33 kD) uPA, tPA, or epidermal growth factor did not compete for binding, demonstrating specificity. Acid treatment, which removed endogenous uPA from the receptor, was required to detect maximal binding (45,000 sites per cell). To investigate the possibility that the uPA receptor on keratinocytes may be involved in epithelial migration during wound repair, cultures were wounded and allowed to migrate into the wounded site. Binding sites for uPA were localized by autoradiographic analysis of 125I-uPA binding as well as by immunocytochemical studies using anti-uPA IgG. With both techniques uPA binding sites were detected selectively on the plasma membrane of cells at the leading edge of the migrating epithelial sheet. This localization pattern suggests that uPA receptor expression on keratinocytes may be coupled to cell migration during cutaneous wounding. Images PMID:1965151

  15. Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer

    SciTech Connect

    Chen, Yi; Pirisi, Lucia; Creek, Kim E.

    2013-09-15

    We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cell proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer. - Highlights: • Ski oncoprotein levels increase during progression of HPV16-transformed cells. • Ski and phospho-Ski protein levels are cell cycle dependent. • Ski knock-down in HPV16-transformed keratinocytes inhibited cell proliferation. • Cervical cancer samples overexpress Ski.

  16. Epiprofin orchestrates epidermal keratinocyte proliferation and differentiation

    PubMed Central

    Nakamura, Takashi; Yoshitomi, Yasuo; Sakai, Kiyoshi; Patel, Vyomesh; Fukumoto, Satoshi; Yamada, Yoshihiko

    2014-01-01

    ABSTRACT The basal layer of the epidermis contains stem cells and transit amplifying cells that rapidly proliferate and differentiate further into the upper layers of the epidermis. A number of molecules have been identified as regulators of this process, including p63 (also known as tumor protein 63) and Notch1. However, little is known about the mechanisms that regulate the transitions from stem cell to proliferating or differentiating transit amplifying cell. Here, we demonstrate that epiprofin (Epfn, also known as Sp6) plays crucial distinct roles in these transition stages as a cell cycle regulator and a transcription factor. Epfn knockout mice have a thickened epidermis, in which p63-expressing basal cells form multiple layers owing to the accumulation of premature transit amplifying cells with reduced proliferation and a reduction in the number of differentiating keratinocytes expressing Notch1. We found that low levels of Epfn expression increased the proliferation of human immortalized keratinocyte (HaCaT) cells by increasing EGF responsiveness and superphosphorylation of Rb. By contrast, high levels of Epfn expression promoted cell cycle exit and differentiation, by reducing E2F transactivation and inducing Notch1 expression. Our findings identify multiple novel functions of Epfn in epidermal development. PMID:25344255

  17. Epiprofin orchestrates epidermal keratinocyte proliferation and differentiation.

    PubMed

    Nakamura, Takashi; Yoshitomi, Yasuo; Sakai, Kiyoshi; Patel, Vyomesh; Fukumoto, Satoshi; Yamada, Yoshihiko

    2014-12-15

    The basal layer of the epidermis contains stem cells and transit amplifying cells that rapidly proliferate and differentiate further into the upper layers of the epidermis. A number of molecules have been identified as regulators of this process, including p63 (also known as tumor protein 63) and Notch1. However, little is known about the mechanisms that regulate the transitions from stem cell to proliferating or differentiating transit amplifying cell. Here, we demonstrate that epiprofin (Epfn, also known as Sp6) plays crucial distinct roles in these transition stages as a cell cycle regulator and a transcription factor. Epfn knockout mice have a thickened epidermis, in which p63-expressing basal cells form multiple layers owing to the accumulation of premature transit amplifying cells with reduced proliferation and a reduction in the number of differentiating keratinocytes expressing Notch1. We found that low levels of Epfn expression increased the proliferation of human immortalized keratinocyte (HaCaT) cells by increasing EGF responsiveness and superphosphorylation of Rb. By contrast, high levels of Epfn expression promoted cell cycle exit and differentiation, by reducing E2F transactivation and inducing Notch1 expression. Our findings identify multiple novel functions of Epfn in epidermal development. PMID:25344255

  18. Comparison of the enzymatic and explant methods for the culture of keratinocytes isolated from human foreskin

    PubMed Central

    ORAZIZADEH, MAHMOUD; HASHEMITABAR, MAHMOUD; BAHRAMZADEH, SOMAYEH; DEHBASHI, FRESHTEH NEJAD; SAREMY, SADEGH

    2015-01-01

    Currently, culture and growth keratinocytes are important stages in achieving a reliable and reproducible skin tissue. In the present study, two different methods, enzymatic and explant methods, for keratinocytes isolation from human foreskin were compared. Foreskins were cut into 2–3 mm pieces and placed in trypsin at 4°C overnight for separation of the epidermis from the dermis. Subsequently, these samples were divided into two groups: i) Keratinocytes separated from the epidermis by trypsin and ii) by the explant method. These keratinocytes were divided into two groups: i) With no feeder layer and ii) onto a type I collagen scaffold. The cells were evaluated using immunocytochemistry and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) staining. In the enzymatic treatment, after 7–10 days no attached cells were found in the cell culture dishes. In the explant method, keratinocytes were separated after ~24 h, attached rapidly and formed big colonies into a collagen scaffold. In the absence of a feeder layer, small colonies were developed with rapid loss of proliferation within 2–3 days. Keratinocytes showed positive immunoreactivity for the pan-cytokeratin marker and keratinocytes' nuclei were clearly observed. This method could be applied and developed as a component of skin substitutes to treat burns and wounds and also in laboratory testing. PMID:26137227

  19. Reduced growth factor requirement of keloid-derived fibroblasts may account for tumor growth

    SciTech Connect

    Russell, S.B.; Trupin, K.M.; Rodriguez-Eaton, S.; Russell, J.D.; Trupin, J.S.

    1988-01-01

    Keloids are benign dermal tumors that form during an abnormal wound-healing process is genetically susceptible individuals. Although growth of normal and keloid cells did not differ in medium containing 10% (vol/vol) fetal bovine serum, keloid culture grew to significantly higher densities than normal cells in medium containing 5% (vol/vol) fetal bovine serum, keloid cultures grew to significantly higher densities than normal cells in medium containing 5% (vol/vol) plasma or 1% fetal bovine serum. Conditioned medium from keloid cultures did not stimulate growth of normal cells in plasma nor did it contain detectable platelet-derived growth factor or epidermal growth factor. Keloid fibroblasts responded differently than normal adult fibroblasts to transforming growth factor ..beta... Whereas transforming growth factor ..beta.. reduced growth stimulation by epidermal growth factor in cells from normal adult skin or scars, it enhanced the activity of epidermal growth factor in cells from normal adult skin or scars, it enhanced the activity of epidermal growth factor in cells from keloids. Normal and keloid fibroblasts also responded differently to hydrocortisone: growth was stimulated in normal adult cells and unaffected or inhibited in keloid cells. Fetal fibroblasts resembled keloid cells in their ability to grow in plasma and in their response to hydrocortisone. The ability of keloid fibroblasts to grow to higher cell densities in low-serum medium than cells from normal adult skin or from normal early or mature scars suggests that a reduced dependence on serum growth factors may account for their prolonged growth in vivo. Similarities between keloid and fetal cells suggest that keloids may result from the untimely expression of growth-control mechanism that is developmentally regulated.

  20. Growth Factors Regulate Expression of Mineral Associated Genes in Cementoblasts

    PubMed Central

    Saygin, N. Esra; Tokiyasu, Yoshihiko; Giannobile, William V.; Somerman, Martha J.

    2008-01-01

    Background Knowledge of the responsiveness of cells within the periodontal region to specific bioactive agents is important for improving regenerative therapies. The aim of this study was to determine the effect of specific growth factors, insulin-like growth factor-I (IGF-I), platelet-derived growth factor-BB (PDGF-BB), and transforming growth factor-β (TGF-β) on cementoblasts in vitro and ex vivo. Methods Osteocalcin (OC) promoter driven SV40 transgenic mice were used to obtain immortalized cementoblasts. Growth factor effects on DNA synthesis were assayed by [3H]-thymidine incorporation. Northern analysis was used to determine the effects of growth factors on gene expression profile. Effects of growth factors on cementoblast induced biomineralization were determined in vitro (von Kossa stain) and ex vivo (re-implantation of cells in immunodeficient (SCID) mice). Results All growth factors stimulated DNA synthesis compared to control. Twenty-four hour exposure of cells to PDGF-BB or TGF-β resulted in a decrease in bone sialoprotein (BSP) and osteocalcin (OCN) mRNAs while PDGF-BB also increased osteopontin (OPN) mRNA. Cells exposed to IGF-I for 24 hours exhibited decreased transcripts for OCN and OPN with an upregulation of BSP mRNA noted at 72 hours. In vitro mineralization was inhibited by continuous application of PDGF-BB or TGF-β, while cells exposed to these factors prior to implantation into SCID mice still promoted biomineralization. Conclusions These data indicate IGF-I, PDGF-BB, and TGF-β influence mitogenesis, phenotypic gene expression profile, and biomineralization potential of cementoblasts suggesting that such factors alone or in combination with other agents may provide trigger factors required for regenerating periodontal tissues. PMID:11063392

  1. High-growth-factor implosions (HEP4)

    SciTech Connect

    Landen, O.L.; Keane, C.J.; Hammel, B.A.

    1996-06-01

    In inertial confinement fusion (ICF), the kinetic energy of an ablating, inward-driven, solid spherical shell is used to compressionally heat the low-density fuel inside. For a given drive, the maximum achievable compressed fuel density and temperature - and hence the maximum neutron production rate depend on the degree of shell isentropy and integrity maintained during the compression. Shell integrity will be degraded by hydrodynamic instability growth of areal density imperfections in the capsule. Surface imperfections on the shell grow as a result of the Richtmyer-Meshkov and Rayleigh-Taylor (RT) instabilities when the shell is accelerated by the ablating lower-density plasma. Perturbations at the outer capsule surface are transferred hydrodynamically to the inner surface, where deceleration of the shell by the lower-density fuel gives rise to further RT growth at the pusher-fuel interface.

  2. Targeting the Insulin Growth Factor and the Vascular Endothelial Growth Factor Pathways in Ovarian Cancer

    PubMed Central

    Shao, Minghai; Hollar, Stacy; Chambliss, Daphne; Schmitt, Jordan; Emerson, Robert; Chelladurai, Bhadrani; Perkins, Susan; Ivan, Mircea; Matei, Daniela

    2015-01-01

    Antiangiogenic therapy is emerging as a highly promising strategy for the treatment of ovarian cancer, but the clinical benefits are usually transitory. The purpose of this study was to identify and target alternative angiogenic pathways that are upregulated in ovarian xenografts during treatment with bevacizumab. For this, angiogenesis-focused gene expression arrays were used to measure gene expression levels in SKOV3 and A2780 serous ovarian xenografts treated with bevacizumab or control. Reverse transcription-PCR was used for results validation. The insulin growth factor 1 (IGF-1) was found upregulated in tumor and stromal cells in the two ovarian xenograft models treated with bevacizumab. Cixutumumab was used to block IGF-1 signaling in vivo. Dual anti-VEGF and IGF blockade with bevacizumab and cixutumumab resulted in increased inhibition of tumor growth. Immunohistochemistry measured multivessel density, Akt activation, and cell proliferation, whereas terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay measured apoptosis in ovarian cancer xenografts. Bevacizumab and cixutumumab combination increased tumor cell apoptosis in vivo compared with therapy targeting either individual pathway. The combination blocked angiogenesis and cell proliferation but not more significantly than each antibody alone. In summary, IGF-1 activation represents an important mechanism of adaptive escape during anti-VEGF therapy in ovarian cancer. This study provides the rationale for designing bevacizumab-based combination regimens to enhance antitumor activity. PMID:22700681

  3. Latent transforming growth factor binding protein 4 regulates transforming growth factor beta receptor stability.

    PubMed

    Su, Chi-Ting; Huang, Jenq-Wen; Chiang, Chih-Kang; Lawrence, Elizabeth C; Levine, Kara L; Dabovic, Branka; Jung, Christine; Davis, Elaine C; Madan-Khetarpal, Suneeta; Urban, Zsolt

    2015-07-15

    Mutations in the gene for the latent transforming growth factor beta binding protein 4 (LTBP4) cause autosomal recessive cutis laxa type 1C. To understand the molecular disease mechanisms of this disease, we investigated the impact of LTBP4 loss on transforming growth factor beta (TGFβ) signaling. Despite elevated extracellular TGFβ activity, downstream signaling molecules of the TGFβ pathway, including pSMAD2 and pERK, were down-regulated in LTBP4 mutant human dermal fibroblasts. In addition, TGFβ receptors 1 and 2 (TGFBR1 and TGFBR2) were reduced at the protein but not at the ribonucleic acid level. Treatment with exogenous TGFβ1 led to an initially rapid increase in SMAD2 phosphorylation followed by a sustained depression of phosphorylation and receptor abundance. In mutant cells TGFBR1 was co-localized with lysosomes. Treatment with a TGFBR1 kinase inhibitor, endocytosis inhibitors or a lysosome inhibitor, normalized the levels of TGFBR1 and TGFBR2. Co-immunoprecipitation demonstrated a molecular interaction between LTBP4 and TGFBR2. Knockdown of LTBP4 reduced TGFβ receptor abundance and signaling in normal cells and supplementation of recombinant LTBP4 enhanced these measures in mutant cells. In a mouse model of Ltbp4 deficiency, reduced TGFβ signaling and receptor levels were normalized upon TGFBR1 kinase inhibitor treatment. Our results show that LTBP4 interacts with TGFBR2 and stabilizes TGFβ receptors by preventing their endocytosis and lysosomal degradation in a ligand-dependent and receptor kinase activity-dependent manner. These findings identify LTBP4 as a key molecule required for the stability of the TGFβ receptor complex, and a new mechanism by which the extracellular matrix regulates cytokine receptor signaling.

  4. Latent transforming growth factor binding protein 4 regulates transforming growth factor beta receptor stability

    PubMed Central

    Su, Chi-Ting; Huang, Jenq-Wen; Chiang, Chih-Kang; Lawrence, Elizabeth C.; Levine, Kara L.; Dabovic, Branka; Jung, Christine; Davis, Elaine C.; Madan-Khetarpal, Suneeta; Urban, Zsolt

    2015-01-01

    Mutations in the gene for the latent transforming growth factor beta binding protein 4 (LTBP4) cause autosomal recessive cutis laxa type 1C. To understand the molecular disease mechanisms of this disease, we investigated the impact of LTBP4 loss on transforming growth factor beta (TGFβ) signaling. Despite elevated extracellular TGFβ activity, downstream signaling molecules of the TGFβ pathway, including pSMAD2 and pERK, were down-regulated in LTBP4 mutant human dermal fibroblasts. In addition, TGFβ receptors 1 and 2 (TGFBR1 and TGFBR2) were reduced at the protein but not at the ribonucleic acid level. Treatment with exogenous TGFβ1 led to an initially rapid increase in SMAD2 phosphorylation followed by a sustained depression of phosphorylation and receptor abundance. In mutant cells TGFBR1 was co-localized with lysosomes. Treatment with a TGFBR1 kinase inhibitor, endocytosis inhibitors or a lysosome inhibitor, normalized the levels of TGFBR1 and TGFBR2. Co-immunoprecipitation demonstrated a molecular interaction between LTBP4 and TGFBR2. Knockdown of LTBP4 reduced TGFβ receptor abundance and signaling in normal cells and supplementation of recombinant LTBP4 enhanced these measures in mutant cells. In a mouse model of Ltbp4 deficiency, reduced TGFβ signaling and receptor levels were normalized upon TGFBR1 kinase inhibitor treatment. Our results show that LTBP4 interacts with TGFBR2 and stabilizes TGFβ receptors by preventing their endocytosis and lysosomal degradation in a ligand-dependent and receptor kinase activity-dependent manner. These findings identify LTBP4 as a key molecule required for the stability of the TGFβ receptor complex, and a new mechanism by which the extracellular matrix regulates cytokine receptor signaling. PMID:25882708

  5. Dual chain synthetic heparin-binding growth factor analogs

    DOEpatents

    Zamora, Paul O.; Pena, Louis A.; Lin, Xinhua

    2009-10-06

    The invention provides synthetic heparin-binding growth factor analogs having two peptide chains each branched from a branch moiety, such as trifunctional amino acid residues, the branch moieties separated by a first linker of from 3 to about 20 backbone atoms, which peptide chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a second linker, which may be a hydrophobic second linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

  6. Dual chain synthetic heparin-binding growth factor analogs

    DOEpatents

    Zamora, Paul O.; Pena, Louis A.; Lin, Xinhua

    2012-04-24

    The invention provides synthetic heparin-binding growth factor analogs having two peptide chains each branched from a branch moiety, such as trifunctional amino acid residues, the branch moieties separated by a first linker of from 3 to about 20 backbone atoms, which peptide chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a second linker, which may be a hydrophobic second linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

  7. Platelet-rich growth factor in oral and maxillofacial surgery

    PubMed Central

    Pal, Uma Shanker; Mohammad, Shadab; Singh, Rakesh K.; Das, Somdipto; Singh, Nimisha; Singh, Mayank

    2012-01-01

    Platelet-rich growth factor is an innovative regenerative therapy used to promote hard and soft tissue healing. It involves the application of autologous platelet-leukocyte-rich plasma containing growth factors and thrombin directly to the site of treatment. It is the intrinsic growth factors released by activated platelets which are concentrated in a topical gel formula. Clinically, it is an affordable treatment with potentially broad spectrum of applications in maxillofacial surgery especially in the treatment of complex or refractory wounds. The present article reviews its various applications not only in the specialization of oral and maxillofacial surgery but also in regenerative medicine. PMID:23833484

  8. Novel biodegradable polymers for local growth factor delivery.

    PubMed

    Amsden, Brian

    2015-11-01

    Growth factors represent an important therapeutic protein drug class, and would benefit significantly from formulations that provide sustained, local release to realize their full clinical potential. Biodegradable polymer-based delivery platforms have been examined to achieve this end; however, formulations based on conventional polymers have yet to yield a clinical product. This review examines new polymer biomaterials that have been developed for growth factor delivery. The dosage forms are discussed in terms of their mechanism of release, the stability of the released growth factor, their method of preparation, and their potential for clinical translation. PMID:26614555

  9. Activation of Nrf2 in keratinocytes causes chloracne (MADISH)-like skin disease in mice

    PubMed Central

    Schäfer, Matthias; Willrodt, Ann-Helen; Kurinna, Svitlana; Link, Andrea S; Farwanah, Hany; Geusau, Alexandra; Gruber, Florian; Sorg, Olivier; Huebner, Aaron J; Roop, Dennis R; Sandhoff, Konrad; Saurat, Jean-Hilaire; Tschachler, Erwin; Schneider, Marlon R; Langbein, Lutz; Bloch, Wilhelm; Beer, Hans-Dietmar; Werner, Sabine

    2014-01-01

    The transcription factor Nrf2 is a key regulator of the cellular stress response, and pharmacological Nrf2 activation is a promising strategy for skin protection and cancer prevention. We show here that prolonged Nrf2 activation in keratinocytes causes sebaceous gland enlargement and seborrhea in mice due to upregulation of the growth factor epigen, which we identified as a novel Nrf2 target. This was accompanied by thickening and hyperkeratosis of hair follicle infundibula. These abnormalities caused dilatation of infundibula, hair loss, and cyst development upon aging. Upregulation of epigen, secretory leukocyte peptidase inhibitor (Slpi), and small proline-rich protein 2d (Sprr2d) in hair follicles was identified as the likely cause of infundibular acanthosis, hyperkeratosis, and cyst formation. These alterations were highly reminiscent to the phenotype of chloracne/“metabolizing acquired dioxin-induced skin hamartomas” (MADISH) patients. Indeed, SLPI, SPRR2, and epigen were strongly expressed in cysts of MADISH patients and upregulated by dioxin in human keratinocytes in an NRF2-dependent manner. These results identify novel Nrf2 activities in the pilosebaceous unit and point to a role of NRF2 in MADISH pathogenesis. PMID:24503019

  10. Role of VEGF Receptors in Normal and Psoriatic Human Keratinocytes: Evidence from Irradiation with Different UV Sources

    PubMed Central

    Zhu, Jian-Wei; Wu, Xian-Jie; Lu, Zhong-Fa; Luo, Dan; Cai, Sui-Qing; Zheng, Min

    2013-01-01

    Vascular endothelial growth factor (VEGF) promotes angiogenesis and plays important roles both in physiological and pathological conditions. VEGF receptors (VEGFRs) are high-affinity receptors for VEGF and are originally considered specific to endothelial cells. We previously reported that VEGFRs were also constitutively expressed in normal human keratinocytes and overexpressed in psoriatic epidermis. In addition, UVB can activate VEGFRs in normal keratinocytes, and the activated VEGFR-2 signaling is involved in the pro-survival mechanism. Here, we show that VEGFRs were also upregulated and activated by UVA in normal human keratinocytes via PKC, and interestingly, both the activated VEGFR-1 and VEGFR-2 protected against UVA-induced cell death. As VEGFRs were over-expressed in psoriatic epidermis, we further investigated whether narrowband UVB (NB-UVB) phototherapy or topical halomethasone monohydrate 0.05% cream could affect their expression. Surprisingly, the over-expressed VEGFRs in psoriatic epidermis were significantly attenuated by both treatments. During NB-UVB therapy, VEGFRs declined first in the basal, and then gradually in the upper psoriatic epidermis. VEGFRs were activated in psoriatic epidermis, their activation was enhanced by NB-UVB, but turned undetectable after whole therapy. This process was quite different from that by halomethasone, in which VEGFRs and phospho-VEGFRs decreased in a gradual, homogeneous manner. Our findings further suggest that UV-induced activation of VEGFRs serves as a pro-survival signal for keratinocytes. In addition, VEGFRs may be involved in the pathological process of psoriasis, and UV phototherapy is effective for psoriasis by directly modulating the expression of VEGFRs. PMID:23383198

  11. Co-stimulation of gastrointestinal tumour cell growth by gastrin, transforming growth factor alpha and insulin like growth factor-I.

    PubMed Central

    Durrant, L. G.; Watson, S. A.; Hall, A.; Morris, D. L.

    1991-01-01

    Epidermal growth factor receptors and insulin like growth factor-I receptors were co-expressed on two gastric and three colorectal tumour cell lines. Previous studies have shown that gastrin receptors were also expressed at a low level or two of these cell lines. Both TGF alpha and IGF-I promoted cell growth in all of the cell lines tested. The cell doubling time of a colorectal cell line was reduced from 48 to 30-34 h. Furthermore the effects of the growth factors were additive. Each growth factor also increased the response of the cells to gastrin, but a combination of both growth factors and gastrin did not further increase growth. PMID:1846553

  12. Therapeutic modulation of growth factors and cytokines in regenerative medicine.

    PubMed

    Ioannidou, Effie

    2006-01-01

    Regeneration that takes place in the human body is limited throughout life. Therefore, when organs are irreparably damaged, they are usually replaced with an artificial device or donor organ. The term "regenerative medicine" covers the restoration or replacement of cells, tissues, and organs. Stem cells play a major role in regenerative medicine by providing the way to repopulate organs damaged by disease. Stem cells have the ability to self renew and to regenerate cells of diverse lineages within the tissue in which they reside. Stem cells could originate from embryos or adult tissues. Growth factors are proteins that may act locally or systemically to affect the growth of cells in several ways. Various cell activities, including division, are influenced by growth factors. Cytokines are a family of low-molecular-weight proteins that are produced by numerous cell types and are responsible for regulating the immune response, inflammation, tissue remodeling and cellular differentiation. Target cells of growth factors and cytokines are mesenchymal, epithelial and endothelial cells. These molecules frequently have overlapping activities and can act in an autocrine or paracrine fashion. A complex network of growth factors and cytokines guides cellular differentiation and regeneration in all organs and tissues. The aim of this paper is to review the role of growth factors and cytokines in different organs or systems and explore their therapeutic application in regenerative medicine. The role of stem cells combined with growth factors and cytokines in the regeneration of vascular and hematopoietic, neural, skeletal, pancreatic, periodontal, and mucosal tissue is reviewed. There is evidence that supports the use of growth factors and cytokines in the treatment of neurological diseases, diabetes, cardiovascular disease, periodontal disease, cancer and its complication, oral mucositis. After solving the ethical issues and establishing clear and reasonable regulations

  13. Dental metal-induced innate reactivity in keratinocytes.

    PubMed

    Rachmawati, Dessy; Buskermolen, Jeroen K; Scheper, Rik J; Gibbs, Susan; von Blomberg, B Mary E; van Hoogstraten, Ingrid M W

    2015-12-25

    Gold, nickel, copper and mercury, i.e. four metals frequently used in dental applications, were explored for their capacity to induce innate immune activation in keratinocytes (KC). Due to their anatomical location the latter epithelial cells are key in primary local irritative responses of skin and mucosa. Fresh foreskin-derived keratinocytes and skin and gingiva KC cell lines were studied for IL-8 release as a most sensitive parameter for NF-kB activation. First, we verified that viral-defense mediating TLR3 is a key innate immune receptor in both skin- and mucosa derived keratinocytes. Second, we found that, in line with our earlier finding that ionized gold can mimic viral dsRNA in triggering TLR3, gold is very effective in KC activation. It would appear that epithelial TLR3 can play a key role in both skin- and mucosa localized irritation reactivities to gold. Subsequently we found that not only gold, but also nickel, copper and mercury salts can activate innate immune reactivity in keratinocytes, although the pathways involved remain unclear. Although current alloys have been optimized for minimal leakage of metal ions, secondary factors such as mechanical friction and acidity may still facilitate such leakage. Subsequently, these metal ions may create local irritation, itching and swelling by triggering innate immune reactions, potentially also facilitating the development of metal specific adaptive immunity.

  14. A short peptide GPIGS promotes proliferation of hair bulb keratinocytes and accelerates hair regrowth in mice.

    PubMed

    Tsuruda, Akinori; Kawano, Yasuhiro; Maekawa, Takaaki; Oka, Syuichi

    2005-03-01

    The aim of this study was to discover a novel agent that promotes hair growth. We carried out a screening test in 298 types of conditioned medium (CM) from cultures of bacteria by using a hair bulb keratinocyte (HBK) growth assay. As a result, we found a HBK growth factor in the CM of Bacillus sp. M18. This HBK growth factor was purified by collecting biologically active fractions in three steps, including HP-20 batch processing, LH-20 chromatography and C18 reverse-phase high-pressure liquid chromatography, and identified as a short peptide GPIGS. GPIGS increased Akt phosphorylation in HBKs. Moreover, the GPIGS-stimulated HBK growth was inhibited by the treatment with LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI-3K). These results suggest that GPIGS promotes HBK growth via the PI-3K/Akt pathway. In addition to in vitro tests, GPIGS was found to accelerate hair regrowth in telogen mice. Our results indicate that GPIGS is a potential agent to promote hair growth.

  15. Vitiligo patient-derived keratinocytes exhibit characteristics of normal wound healing via epithelial to mesenchymal transition.

    PubMed

    Banerjee, Poulomi; Venkatachalam, Sandhyaa; Mamidi, Murali Krishna; Bhonde, Ramesh; Shankar, Krupa; Pal, Rajarshi

    2015-05-01

    Vitiligo is an autoimmune disorder that leads to depigmentation of skin via melanocyte dysfunction. Keratinocyte-induced toxicity is one among the several etiological factors implicated for vitiligo, and hence, autologous keratinocyte grafting is projected as one of the primary mode of treatment for vitiligo. However, reports indicate that perilesional keratinocytes not only display signatures of apoptosis but also could secrete cytokines and mediators which have antagonistic effect on proliferation or survival. Therefore, we investigated how vitiligo patients' derived keratinocytes respond to surplus amounts of inflammatory cytokines and whether they recapitulate events that take place during conventional wound healing. The primary objective of our study was to determine whether keratinocytes isolated from a vitiligo patient would undergo epithelial-mesenchymal transition similar to their normal counterparts upon induction with inflammatory cytokines such as TGF-b1 and EGF. We found that these keratinocytes undergo EMT during wound repair accompanied with increase in the levels of mesenchymal markers and ECM proteins; decrease in the levels of epithelial markers and enhanced migratory ability. Besides, we also demonstrated that EMT induction leads to activation of SMAD and MAPK pathways via Ras, Raf, PAI 1, Snail, Slug and ZO1. To our knowledge, this is the first report on the characterization of primary keratinocytes isolated from vitiligo patients with respect to their wound healing capacity.

  16. Release kinetics of platelet-derived and plasma-derived growth factors from autologous plasma rich in growth factors.

    PubMed

    Anitua, Eduardo; Zalduendo, Mari Mar; Alkhraisat, Mohammad Hamdan; Orive, Gorka

    2013-10-01

    Many studies have evaluated the biological effects of platelet rich plasma reporting the final outcomes on cell and tissues. However, few studies have dealt with the kinetics of growth factor delivery by plasma rich in growth factors. Venous blood was obtained from three healthy volunteers and processed with PRGF-Endoret technology to prepare autologous plasma rich in growth factors. The gel-like fibrin scaffolds were then incubated in triplicate, in a cell culture medium to monitor the release of PDGF-AB, VEGF, HGF and IGF-I during 8 days of incubation. A leukocyte-platelet rich plasma was prepared employing the same technology and the concentrations of growth factors and interleukin-1β were determined after 24h of incubation. After each period, the medium was collected, fibrin clot was destroyed and the supernatants were stored at -80°C until analysis. The growth factor delivery is diffusion controlled with a rapid initial release by 30% of the bioactive content after 1h of incubation and a steady state release when almost 70% of the growth factor content has been delivered. Autologous fibrin matrix retained almost 30% of the amount of the growth factors after 8 days of incubation. The addition of leukocytes to the formula of platelet rich plasma did not increase the concentration of the growth factors, while it drastically increased the presence of pro-inflammatory IL-1β. Further studies employing an in vitro inflammatory model would be interesting to study the difference in growth factors and pro-inflammatory cytokines between leukocyte-free and leukocyte-rich platelet rich plasma.

  17. Visualization of growth factor receptor sites in rat forebrain

    SciTech Connect

    Quirion, R.; Araujo, D.; Nair, N.P.; Chabot, J.G.

    1988-01-01

    It is now known that various growth factors may also act in the central nervous system. Among them, it has recently been shown that epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) may possess trophic effects in the mammalian brain. We report here on the respective autoradiographic distribution of (/sup 125/I)EGF and (/sup 125/I)IGF-I receptor binding sites in the rat brain, both during ontogeny and in adulthood. It appears that (/sup 125/I)EGF sites are mostly found in the rat forebrain during brain development. On the other hand, (/sup 125/I)IGF-I sites are more widely distributed both during ontogeny and in adulthood. These results reveal the plasticity of the expression of EGF and IGF-I receptor sites in the mammalian brain. This could be relevant for the respective role of these two growth factors in the development and maintenance of neuronal function.

  18. Abnormal Growth Factor/Cytokine Network in Gastric Cancer

    PubMed Central

    2008-01-01

    Gastric cancer cells express a broad spectrum of the growth factor/cytokine receptor systems that organize the complex interaction between cancer cells and stromal cells in tumor microenvironment, which confers cell growth, apoptosis, morphogenesis, angiogenesis, progression and metastasis. However, these abnormal growth factor/cytokine networks differ in the two histological types of gastric cancer. Importantly, activation of nuclear factor-kB pathway by Helicobacter pylori infection may act as a key player for induction of growth factor/cytokine networks in gastritis and pathogenesis of gastric cancer. Better understanding of these events will no doubt provide new approaches for biomarkers of diagnosis and effective therapeutic targeting of gastric cancer. PMID:19308687

  19. Cardiac Regeneration using Growth Factors: Advances and Challenges

    PubMed Central

    Rebouças, Juliana de Souza; Santos-Magalhães, Nereide Stela; Formiga, Fabio Rocha

    2016-01-01

    Myocardial infarction is the most significant manifestation of ischemic heart disease and is associated with high morbidity and mortality. Novel strategies targeting at regenerating the injured myocardium have been investigated, including gene therapy, cell therapy, and the use of growth factors. Growth factor therapy has aroused interest in cardiovascular medicine because of the regeneration mechanisms induced by these biomolecules, including angiogenesis, extracellular matrix remodeling, cardiomyocyte proliferation, stem-cell recruitment, and others. Together, these mechanisms promote myocardial repair and improvement of the cardiac function. This review aims to address the strategic role of growth factor therapy in cardiac regeneration, considering its innovative and multifactorial character in myocardial repair after ischemic injury. Different issues will be discussed, with emphasis on the regeneration mechanisms as a potential therapeutic resource mediated by growth factors, and the challenges to make these proteins therapeutically viable in the field of cardiology and regenerative medicine. PMID:27355588

  20. Activation of protein phosphatase 2A is responsible for increased content and inactivation of respiratory chain complex i induced by all-trans retinoic acid in human keratinocytes.

    PubMed

    Papa, F; Sardaro, N; Lippolis, R; Panelli, D; Scacco, S

    2016-01-01

    This study presents the effect of all-trans retinoic acid (ATRA) on cell growth and respiratory chain complex I in human keratinocyte cultures. Keratinocyte treatment results in increased level of GRIM-19 and other subunits of complex I, in particular of their carbonylated forms, associated with inhibition of its enzymatic activity. The results show that in keratinocytes ATRA-promoted phosphatase activity controls the proteostasis and activity of complex I. PMID:27358125

  1. Brefeldin A reduces tumor necrosis factor-α-stimulated production of inflammatory mediators by suppressing the Akt, mTOR, and NF-κB pathways in human keratinocytes.

    PubMed

    Nam, Yoon Jeong; Lee, Chung Soo

    2016-09-01

    Keratinocytes may play an important role in the pathogenesis of inflammatory skin diseases. Brefeldin A has been shown to attenuate the production and secretion of chemical mediators involved in inflammation and immune responses. However, the effect of brefeldin A on the TNF-α-stimulated production of inflammatory mediators in keratinocytes has not been studied. We investigated the effect of brefeldin A on the TNF-α-stimulated production of inflammatory mediators using HaCaT cells and primary keratinocytes in relation to the Akt, mTOR, and NF-κB pathways, which regulates the transcription genes involved in immune and inflammatory responses. Brefeldin A, Akt inhibitor, Bay 11-7085 (an inhibitor of NF-κB activation), and rapamycin (mTOR inhibitor) inhibited the TNF-α-stimulated productions of inflammatory mediators, and activations of Akt, mTOR, and NF-κB in keratinocytes. The results show that brefeldin A appears to attenuate TNF-α-stimulated inflammatory mediator production in keratinocytes by suppressing the activation of the Akt, mTOR, and NF-κB pathways. PMID:27198515

  2. Regulation of human amnion cell growth and morphology by sera, plasma, and growth factors.

    PubMed

    Gaffney, E V; Grimaldi, M A

    1981-01-01

    The requirements of human epithelial cells derived from the amnion membrane for serum factors were investigated. The growth promoting effects of human whole blood serum (WBS), platelet-poor defibrinogenated plasma, and plasma-derived serum (PDS) were examined in primary cultures of these ectodermal cells. The numbers of population doublings recorded after 10 days in the presence of 10% WBS, defibrinogenated plasma, or PDS were 2.3, 2.0 or 1.5, respectively. Although dialysis of sera or plasma had little effect on growth promotion, it markedly decreased the capacity of plasma to maintain cells in culture beyond 10 days. The differences in growth activities could not be attributed to the presence of anticoagulant in plasma and PDS or to the presence of excess calcium in PDS. Platelet lysates and purified platelet-derived growth factor had no effect on growth. Amnion cell growth was enhanced by epidermal growth factor (EGF) or hydrocortisone, but the glucocorticoid did not condition cells to respond to growth factors. Insulin and fibroblast growth factor singly or in combination had no effect on cell replication. Giant cell formation accompanied maintenance in hydrocortisone with defibrinogenated plasma and PDS. Discrete regions of dense population appeared in the presence of hydrocortisone, EGF, and undialyzed supplements.

  3. Lifetime growth in wild meerkats: incorporating life history and environmental factors into a standard growth model.

    PubMed

    English, Sinéad; Bateman, Andrew W; Clutton-Brock, Tim H

    2012-05-01

    Lifetime records of changes in individual size or mass in wild animals are scarce and, as such, few studies have attempted to model variation in these traits across the lifespan or to assess the factors that affect them. However, quantifying lifetime growth is essential for understanding trade-offs between growth and other life history parameters, such as reproductive performance or survival. Here, we used model selection based on information theory to measure changes in body mass over the lifespan of wild meerkats, and compared the relative fits of several standard growth models (monomolecular, von Bertalanffy, Gompertz, logistic and Richards). We found that meerkats exhibit monomolecular growth, with the best model incorporating separate growth rates before and after nutritional independence, as well as effects of season and total rainfall in the previous nine months. Our study demonstrates how simple growth curves may be improved by considering life history and environmental factors, which may be particularly relevant when quantifying growth patterns in wild populations.

  4. Endothelin-1 is a transcriptional target of p53 in epidermal keratinocytes and regulates ultraviolet-induced melanocyte homeostasis.

    PubMed

    Hyter, Stephen; Coleman, Daniel J; Ganguli-Indra, Gitali; Merrill, Gary F; Ma, Steven; Yanagisawa, Masashi; Indra, Arup K

    2013-03-01

    Keratinocytes contribute to melanocyte activity by influencing their microenvironment, in part, through secretion of paracrine factors. Here, we discovered that p53 directly regulates Edn1 expression in epidermal keratinocytes and controls UV-induced melanocyte homeostasis. Selective ablation of endothelin-1 (EDN1) in murine epidermis (EDN1(ep-/-) ) does not alter melanocyte homeostasis in newborn skin but decreases dermal melanocytes in adult skin. Results showed that keratinocytic EDN1 in a non-cell autonomous manner controls melanocyte proliferation, migration, DNA damage, and apoptosis after ultraviolet B (UVB) irradiation. Expression of other keratinocyte-derived paracrine factors did not compensate for the loss of EDN1. Topical treatment with EDN1 receptor (EDNRB) antagonist BQ788 abrogated UV-induced melanocyte activation and recapitulated the phenotype seen in EDN1(ep-/-) mice. Altogether, the present studies establish an essential role of EDN1 in epidermal keratinocytes to mediate UV-induced melanocyte homeostasis in vivo.

  5. Regulation of Srpr Expression by miR-330-5p Controls Proliferation of Mouse Epidermal Keratinocyte

    PubMed Central

    Kim, Bong-Kyu; Yoo, Hye-In; Choi, Keonwoo; Lee, Ah-Reum; Yoon, Sungjoo Kim

    2016-01-01

    Srpr is a gene encoding α subunit of the signal recognition particle receptor which is involved in the targeting and translocation of nascent secretory and membrane proteins to the endoplasmic reticulum. Previous studies showed aberrant expression of Srpr in several cell types with abnormal growth rate. Although Srpr is expressed in various tissues including skin, the role of Srpr in keratinocytes and regulation of its expression by miRNAs have not been studied. In this study, we investigated the role of SRPR and regulation of its expression by miRNA in skin keratinocytes. We found that SRPR was highly expressed in epidermal keratinocytes and regulated keratinocyte proliferation by affecting cell cycle progression. We also demonstrated that miR-330-5p directly inhibits Srpr expression. These data suggest that miR-330-5p-mediated regulation of the SRPR level is needed for the regulation of proliferation of epidermal keratinocytes. PMID:27768721

  6. Distribution of insulin-like growth factors in condylar hyperplasia.

    PubMed

    Götz, Werner; Lehmann, Tim Sebastian; Appel, Thorsten Robin; Rath-Deschner, Birgit; Dettmeyer, Reinhard; Luder, Hans-Ulrich; Reich, Rudolf H; Jäger, Andreas

    2007-01-01

    Condylar hyperplasia (CH) is a local overgrowth of the condylar process of the temporomandibular joint (TMJ) of unknown etiology. Probably, growth factors like the insulin-like growth factors (IGFs) are involved in its pathogenesis. Specimens from 12 patients were investigated histologically and immunohistochemically to obtain the distribution of the IGFs-I and -II and the IGF1 receptor. The results revealed juvenile and adult subtypes. While generally IGF-II could only be detected weakly, in the juvenile cases strong immunostaining for IGF-I in cartilage and bone supposes an influence on pathological growth processes. PMID:17695990

  7. Nerve growth factor binding domain of the nerve growth factor receptor

    SciTech Connect

    Welcher, A.A.; Bitler, C.M.; Radeke, M.J.; Shooter, E.M. )

    1991-01-01

    A structural analysis of the rat low-affinity nerve growth factor (NGF) receptor was undertaken to define the NGF binding domain. Mutant NGF receptor DNA constructs were expressed in mouse fibroblasts or COS cells, and the ability of the mutant receptors to bind NGF was assayed. In the first mutant, all but 16 amino acid residues of the intracellular domain of the receptor were removed. This receptor bound NGF with a K{sub d} comparable to that of the wild-type receptor. A second mutant contained only the four cysteine-rich sequences from the extracellular portion of the protein. This mutant was expressed in COS cells and the resultant protein was a secreted soluble form of the receptor that was able to bind NGF. Two N-terminal deletions, in which either the first cystein-rich sequence or the first and part of the second cystein-rich sequences were removed, bound NGF. However, a mutant lacking all four cysteine-rich sequences was unable to bind NGF. These results show that the four cysteine-rich sequences of the NGF receptor contain the NGF binding domain.

  8. [Novel role of growth factors in ovary function].

    PubMed

    Amsterdam, Abraham

    2010-12-01

    The development of the DNA microarray technique facilitated systematic studies of the modulation of gene function. Considerable attention has been focused on members of the growth factor family to elucidate the main regulators of oocyte maturation and ovarian follicle rupture. Among these growth factors, it was found, both in rodents and in humans, that amphiregulin (Ar) and epiregulin (Ep) of the epidermal growth factor (EGF) family were dramatically up-regulated by gonadotrophins in the intact ovary and in primary granulosa cells, respectively. Their role in cumulus expansion and oocyte maturation was established in rodents, and their synthesis under LH stimulation in granulosa cells was demonstrated in humans. To be activated, Ar and Ep must be cleaved by a disintegrin and metalloproteinases (ADAMs) family. However, the precise processing of Ar and Ep by the cumulus cells is still obscure. Future investigations using DNA microarray technique may reveal the repertoire of genes activated in Ar- and Ep-stimulated cumulus cells and may help elucidate the molecular basis of ovulation. EFG-like factors are also involved in triggering ovarian cancer The author hypothesized that the normal ovary maintains cyclicity in the formation of these growth factors preventing the ovary from developing ovarian cancer In ovarian cancer these growth factors are continuously formed in an autocrine manner, leading to transformation and subsequently to ovarian cancer. These growth factors are essential for both normal and neoplastic transformation of the ovary. Taking into consideration these growth factors in the treatment of ovarian malfunction may be one way of curing ovarian cancer. PMID:21916103

  9. Cysteine Prevents the Reduction in Keratin Synthesis Induced by Iron Deficiency in Human Keratinocytes.

    PubMed

    Miniaci, Maria Concetta; Irace, Carlo; Capuozzo, Antonella; Piccolo, Marialuisa; Di Pascale, Antonio; Russo, Annapina; Lippiello, Pellegrino; Lepre, Fabio; Russo, Giulia; Santamaria, Rita

    2016-02-01

    L-cysteine is currently recognized as a conditionally essential sulphur amino acid. Besides contributing to many biological pathways, cysteine is a key component of the keratin protein by its ability to form disulfide bridges that confer strength and rigidity to the protein. In addition to cysteine, iron represents another critical factor in regulating keratins expression in epidermal tissues, as well as in hair follicle growth and maturation. By focusing on human keratinocytes, the aim of this study was to evaluate the effect of cysteine supplementation as nutraceutical on keratin biosynthesis, as well as to get an insight on the interplay of cysteine availability and cellular iron status in regulating keratins expression in vitro. Herein we demonstrate that cysteine promotes a significant up-regulation of keratins expression as a result of de novo protein synthesis, while the lack of iron impairs keratin expression. Interestingly, cysteine supplementation counteracts the adverse effect of iron deficiency on cellular keratin expression. This effect was likely mediated by the up-regulation of transferrin receptor and ferritin, the main cellular proteins involved in iron homeostasis, at last affecting the labile iron pool. In this manner, cysteine may also enhance the metabolic iron availability for DNA synthesis without creating a detrimental condition of iron overload. To the best of our knowledge, this is one of the first study in an in vitro keratinocyte model providing evidence that cysteine and iron cooperate for keratins expression, indicative of their central role in maintaining healthy epithelia.

  10. Insulin-like 3-induced rat preantral follicular growth is mediated by growth differentiation factor 9.

    PubMed

    Xue, Kai; Kim, Ji Young; Liu, Jia-yin; Tsang, Benjamin K

    2014-01-01

    The communication of somatic cells and oocytes by intrafollicular paracrine factors is essential for follicular growth in the ovary. Insulin-like 3 (INSL3) is a theca cell-secreted paracrine factor. Androgens and growth differentiation factor 9 (GDF9), an oocyte-derived growth factor, are essential for follicular development. Using a rat preantral follicle culture model, we examined in the present study the influence of INSL3 on preantral follicular growth and the molecular mechanisms involved. We have observed that the receptor for INSL3, relaxin/insulin-like family peptide receptor 2 (RXFP2), was exclusively expressed in oocytes. Recombinant INSL3 stimulated Gdf9 expression, preantral follicular growth, and testosterone synthesis in vitro. Inhibition of the cAMP/protein kinase A signaling pathway (with cAMP antagonist, 8-bromoadenosine 3',5'-cyclic monophosphorothioate, Rp-isomer) attenuated INSL3-induced Gdf9 expression and preantral follicular growth. Moreover, knocking down Gdf9 expression (with small interfering RNA) or inhibiting GDF9 signaling (with SB431542, an activin receptor-like kinase receptor 5 inhibitor, or specific inhibitor of mothers against decapentaplegic homolog 3) or androgen action (with flutamide, an androgen receptor antagonist) suppressed INSL3-induced preantral follicular growth. In addition, LH and DHT regulated the expression of Insl3 mRNA in preantral follicles. These observations suggest that INSL3 is a key theca cell-derived growth factor for preantral follicle and that its action is mediated by GDF9.

  11. A high yield method for growing primary canine keratinocytes.

    PubMed

    Watson, Adrian; Baker, Claire; Bailey, Julie; Fray, Tim; Markwell, Peter

    2004-07-01

    From a small amount of starting material, a large quantity of canine keratinocytes can be generated for experimental purposes using a refined method of explant culture to initiate the growth of basal cells with a high proliferative potential. The dividing capacity of cultures was promoted by a system selecting clonogenic cells onto an i3T3 feeder layer in combination with carefully monitoring cell morphology and passaging to select out excessive numbers of differentiated keratinocytes. Levels of contaminating dermal fibroblasts, which if left unchecked will overgrow keratinocytes, were kept to a minimum by a combination of careful explant micro-dissection to remove dermis, eliminating explants with signs of fibroblast growth as well as using cholera toxin, EGF and i3T3 feeder layers. The advantage of the method described is that it does not rely on the provision of large quantities of starting material thereby reducing the need for repeated tissue sampling, and passage numbers of five or six can be routinely achieved. This technique can therefore be useful to experimenters who require a regular and reliable source of cells for their studies. PMID:15158212

  12. Cutaneous adverse reactions specific to epidermal growth factor receptor inhibitors

    PubMed Central

    Lupu, I; Voiculescu, VM; Bacalbasa, N; Prie, BE; Cojocaru, I; Giurcaneanu, C

    2015-01-01

    Classical antineoplastic therapy is encumbered by extensively studied adverse reactions, most often of systemic nature. The emergence of new generations of anticancer treatments, including epidermal growth factor receptor inhibitors, besides improving the response to treatment and the survival rate, is accompanied by the occurrence of new specific side effects, incompletely studied. These side effects are most often cutaneous (hand foot syndrome, acneiform reactions), and in some cases are extremely severe, requiring dose reduction or drug discontinuation. The prevention of the cutaneous adverse effects and their treatment require a close collaboration between the oncologist and the dermatologist. The occurrence of some of these skin adverse effects may be a favorable prognostic factor for the response to the cancer treatment and the overall survival. Abbreviations: EGFR = epidermal growth factor receptors; EGFRI = epidermal growth factor receptors inhibitors PMID:26361513

  13. Exogenous stimulation with Eclipta alba promotes hair matrix keratinocyte proliferation and downregulates TGF-β1 expression in nude mice.

    PubMed

    Begum, Shahnaz; Lee, Mi Ra; Gu, Li Juan; Hossain, Jamil; Sung, Chang Keun

    2015-02-01

    Eclipta alba (L.) Hassk (E. alba) is a traditionally acclaimed medicinal herb used for the promotion of hair growth. However, to the best of our knowledge, no report has been issued to date on its effects on genetically distorted hair follicles (HFs). In this study, we aimed to identify an agent (stimuli) that may be beneficial for the restoration of human hair loss and which may be used as an alternative to synthetic drugs. We investigated the effects of petroleum ether extract (PEE) and different solvent fractions of E. alba on HFs of nude mice. Treatment was performed by topical application on the backs of nude mice and the changes in hair growth patterns were evaluated. Histological analysis was carried out to evaluate the HF morphology and the structural differences. Immunohistochemical (IHC) staining was performed to visualize follicular keratinocyte proliferation. The histological assessments revealed that the PEE-treated skin specimens exhibited prominent follicular hypertrophy. Subsequently, IHC staining revealed a significant increase (p<0.001) in the number of follicular keratinocytes in basal epidermal and matrix cells. Our results also demonstrated that PEE significantly (p<0.001) reduced the levels of transforming growth factor-β1 (TGF-β1) expression during early anagen and anagen-catagen transition. Our results suggest that PEE of E. alba acts as an important exogenous mediator that stimulates follicular keratinocyte proliferation and delays terminal differentiation by downregulating TGF-β1 expression. Thus, this study highlights the potential use of PEE of E. alba in the treatment of certain types of alopecia. PMID:25484129

  14. Hepatocyte growth factor, hepatocyte growth factor activator and arginine in a rat fulminant colitis model

    PubMed Central

    Zwintscher, Nathan P.; Shah, Puja M.; Salgar, Shashikumar K.; Newton, Christopher R.; Maykel, Justin A.; Samy, Ahmed; Jabir, Murad; Steele, Scott R.

    2016-01-01

    Introduction Dextran sodium sulfate (DSS) is commonly used to induce a murine fulminant colitis model. Hepatocyte growth factor (HGF) has been shown to decrease the symptoms of inflammatory bowel disease (IBD) but the effect of its activator, HGFA, is not well characterized. Arginine reduces effects of oxidative stress but its effect on IBD is not well known. The primary aim is to determine whether HGF and HGFA, or arginine will decrease IBD symptoms such as pain and diarrhea in a DSS-induced fulminant colitis murine model. Methods A severe colitis was induced in young, male Fischer 344 rats with 4% (w/v) DSS oral solution for seven days; rats were sacrificed on day 10. Rats were divided into five groups of 8 animals: control, HGF (700 mcg/kg/dose), HGF and HGFA (10 mcg/dose), HGF and arginine, and high dose HGF (2800 mcg/kg/dose). Main clinical outcomes were pain, diarrhea and weight loss. Blinded pathologists scored the terminal ileum and distal colon. Results DSS reliably induced severe active colitis in 90% of animals (n = 36/40). There were no differences in injury scores between control and treatment animals. HGF led to 1.38 fewer days in pain (p = 0.036), while arginine led to 1.88 fewer days of diarrhea (P = 0.017) compared to controls. 88% of HGFA-treated rats started regaining weight (P < 0.001). Discussion/Conclusion Although treatment was unable to reverse fulminant disease, HGF and arginine were associated with decreased days of pain and diarrhea. These clinical interventions may reduce associated symptoms for severe IBD patients, even when urgent surgical intervention remains the only viable option. PMID:27144006

  15. Nicotinic α7 receptor inhibits the acylation stimulating protein-induced production of monocyte chemoattractant protein-1 and keratinocyte-derived chemokine in adipocytes by modulating the p38 kinase and nuclear factor-κB signaling pathways

    PubMed Central

    Jiao, Zhou-Yang; Wu, Jing; Liu, Chao; Wen, Bing; Zhao, Wen-Zeng; Du, Xin-Ling

    2016-01-01

    Obesity is associated with chronic low-grade inflammation, which is characterized by increased infiltration of macrophages into adipose tissue. Acylation stimulating protein (ASP) is an adipokine derived from the immune complement system, which constitutes a link between adipocytes and macrophages, and is involved in energy homeostasis and inflammation. The purpose of the present study was to preliminarily investigate in vitro, whether functional α7nAChR in adipocytes may suppress ASP-induced inflammation and determine the possible signaling mechanism. Studies have reported associations between the expression of α7 nicotinic acetylcholine receptor (α7nAChR) and obesity, insulin resistance and diabetes. Additionally, α7nAChRs are important peripheral mediators of chronic inflammation, which is a key contributor to health problems in obesity. The primary aim of the present study was to evaluate the impact of exogenous ASP and α7nAChR on macrophage infiltration in adipose tissue and to examine the potential underlying molecular mechanism. Western blot analysis revealed that recombinant ASP increased the expression levels of monocyte chemoattractant protein-1 (MCP-1) and keratinocyte-derived chemokine (KC) by 3T3-L1 adipocytes. However, nicotine significantly inhibited the production of ASP-induced cytokines via the stimulation of α7nAChR. It was also found that α7nAChR inhibited the ASP-induced activation of p38 kinase and nuclear factor-κB (NF-κB), and the production of MCP-1 and KC. These data indicated that α7nAChR caused the inhibition of ASP-induced activation of p38 kinase and NF-κB to inhibit the production of MCP-1 and KC. PMID:27572255

  16. Beclin 1 regulates growth factor receptor signaling in breast cancer.

    PubMed

    Rohatgi, R A; Janusis, J; Leonard, D; Bellvé, K D; Fogarty, K E; Baehrecke, E H; Corvera, S; Shaw, L M

    2015-10-16

    Beclin 1 is a haploinsufficient tumor suppressor that is decreased in many human tumors. The function of beclin 1 in cancer has been attributed primarily to its role in the degradative process of macroautophagy. However, beclin 1 is a core component of the vacuolar protein sorting 34 (Vps34)/class III phosphatidylinositoI-3 kinase (PI3KC3) and Vps15/p150 complex that regulates multiple membrane-trafficking events. In the current study, we describe an alternative mechanism of action for beclin 1 in breast cancer involving its control of growth factor receptor signaling. We identify a specific stage of early endosome maturation that is regulated by beclin 1, the transition of APPL1-containing phosphatidyIinositol 3-phosphate-negative (PI3P(-)) endosomes to PI3P(+) endosomes. Beclin 1 regulates PI3P production in response to growth factor stimulation to control the residency time of growth factor receptors in the PI3P(-)/APPL(+)-signaling-competent compartment. As a result, suppression of BECN1 sustains growth factor-stimulated AKT and ERK activation resulting in increased breast carcinoma cell invasion. In human breast tumors, beclin 1 expression is inversely correlated with AKT and ERK phosphorylation. Our data identify a novel role for beclin 1 in regulating growth factor signaling and reveal a mechanism by which loss of beclin 1 expression would enhance breast cancer progression.

  17. Cytokine and Growth Factor Responses After Radiotherapy for Localized Ependymoma

    SciTech Connect

    Merchant, Thomas E. Li Chenghong; Xiong Xiaoping; Gaber, M. Waleed

    2009-05-01

    Purpose: To determine the time course and clinical significance of cytokines and peptide growth factors in pediatric patients with ependymoma treated with postoperative radiotherapy (RT). Methods and Materials: We measured 15 cytokines and growth factors (fibroblast growth factor, epidermal growth factor, vascular endothelial growth factor [VEGF], interleukin [IL]-1{beta}, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, interferon-{gamma}, tumor necrosis factor-{alpha}, granulocyte-macrophage colony-stimulating factor, monocyte chemoattractant protein-1, and macrophage inflammatory protein-{alpha}) from 30 patients before RT and 2 and 24 h, weekly for 6 weeks, and at 3, 6, 9, and 12 months after the initiation of RT. Two longitudinal models for the trend of log-transformed measurements were fitted, one during treatment and one through 12 months. Results: During RT, log IL-8 declined at a rate of -0.10389/wk (p = 0.0068). The rate of decline was greater (p = 0.028) for patients with an infratentorial tumor location. The decline in IL-8 after RT was significant when stratified by infratentorial tumor location (p = 0.0345) and more than one surgical procedure (p = 0.0272). During RT, the decline in log VEGF was significant when stratified by the presence of a ventriculoperitoneal shunt. After RT, the log VEGF declined significantly at a rate of -0.06207/mo. The decline was significant for males (p = 0.0222), supratentorial tumors (p = 0.0158), one surgical procedure (p = 0.0222), no ventriculoperitoneal shunt (p = 0.0005), and the absence of treatment failure (p = 0.0028). Conclusion: The pro-inflammatory cytokine IL-8 declined significantly during RT and the decline differed according to tumor location. The angiogenesis factor VEGF declined significantly during the 12 months after RT. The decline was greater in males, those without a ventriculoperitoneal shunt, and in those with favorable disease factors, including one surgical procedure, supratentorial tumor location, and

  18. FOXM1 regulates proliferation, senescence and oxidative stress in keratinocytes and cancer cells

    PubMed Central

    Smirnov, Artem; Panatta, Emanuele; Lena, AnnaMaria; Castiglia, Daniele; Di Daniele, Nicola; Melino, Gerry; Candi, Eleonora

    2016-01-01

    Several transcription factors, including the master regulator of the epidermis, p63, are involved in controlling human keratinocyte proliferation and differentiation. Here, we report that in normal keratinocytes, the expression of FOXM1, a member of the Forkhead superfamily of transcription factors, is controlled by p63. We observe that, together with p63, FOXM1 strongly contributes to the maintenance of high proliferative potential in keratinocytes, whereas its expression decreases during differentiation, as well as during replicative-induced senescence. Depletion of FOXM1 is sufficient to induce keratinocyte senescence, paralleled by an increased ROS production and an inhibition of ROS-scavenger genes (SOD2, CAT, GPX2, PRDX). Interestingly, FOXM1 expression is strongly reduced in keratinocytes isolated from old human subjects compared with young subjects. FOXM1 depletion sensitizes both normal keratinocytes and squamous carcinoma cells to apoptosis and ROS-induced apoptosis. Together, these data identify FOXM1 as a key regulator of ROS in normal dividing epithelial cells and suggest that squamous carcinoma cells may also use FOXM1 to control oxidative stress to escape premature senescence and apoptosis. PMID:27385468

  19. The Retinoid-Related Orphan Receptor RORα Promotes Keratinocyte Differentiation via FOXN1

    PubMed Central

    Dai, Jun; Brooks, Yang; Lefort, Karine; Getsios, Spiro; Dotto, G. Paolo

    2013-01-01

    RORα is a retinoid-related orphan nuclear receptor that regulates inflammation, lipid metabolism, and cellular differentiation of several non-epithelial tissues. In spite of its high expression in skin epithelium, its functions in this tissue remain unclear. Using gain- and loss-of-function approaches to alter RORα gene expression in human keratinocytes (HKCs), we have found that this transcription factor functions as a regulator of epidermal differentiation. Among the 4 RORα isoforms, RORα4 is prominently expressed by keratinocytes in a manner that increases with differentiation. In contrast, RORα levels are significantly lower in skin squamous cell carcinoma tumors (SCCs) and cell lines. Increasing the levels of RORα4 in HKCs enhanced the expression of structural proteins associated with early and late differentiation, as well as genes involved in lipid barrier formation. Gene silencing of RORα impaired the ability of keratinocytes to differentiate in an in vivo epidermal cyst model. The pro-differentiation function of RORα is mediated at least in part by FOXN1, a well-known pro-differentiation transcription factor that we establish as a novel direct target of RORα in keratinocytes. Our results point to RORα as a novel node in the keratinocyte differentiation network and further suggest that the identification of RORα ligands may prove useful for treating skin disorders that are associated with abnormal keratinocyte differentiation, including cancer. PMID:23922987

  20. Role of taurine accumulation in keratinocyte hydration.

    PubMed

    Janeke, Guido; Siefken, Wilfried; Carstensen, Stefanie; Springmann, Gunja; Bleck, Oliver; Steinhart, Hans; Höger, Peter; Wittern, Klaus-Peter; Wenck, Horst; Stäb, Franz; Sauermann, Gerhard; Schreiner, Volker; Doering, Thomas

    2003-08-01

    Epidermal keratinocytes are exposed to a low water concentration at the stratum corneum-stratum granulosum interface. When epithelial tissues are osmotically perturbed, cellular protection and cell volume regulation is mediated by accumulation of organic osmolytes such as taurine. Previous studies reported the presence of taurine in the epidermis of several animal species. Therefore, we analyzed human skin for the presence of the taurine transporter (TAUT) and studied the accumulation of taurine as one potential mechanism protecting epidermal keratinocytes from dehydration. According to our results, TAUT is expressed as a 69 kDa protein in human epidermis but not in the dermis. For the epidermis a gradient was evident with maximal levels of TAUT in the outermost granular keratinocyte layer and lower levels in the stratum spinosum. No TAUT was found in the basal layer or in the stratum corneum. Keratinocyte accumulation of taurine was induced by experimental induction of skin dryness via application of silica gel to human skin. Cultured human keratinocytes accumulated taurine in a concentration- and osmolarity-dependent manner. TAUT mRNA levels were increased after exposure of human keratinocytes to hyperosmotic culture medium, indicating osmosensitive TAUT mRNA expression as part of the adaptation of keratinocytes to hyperosmotic stress. Keratinocyte uptake of taurine was inhibited by beta-alanine but not by other osmolytes such as betaine, inositol, or sorbitol. Accumulation of taurine protected cultured human keratinocytes from both osmotically induced and ultraviolet-induced apoptosis. Our data indicate that taurine is an important epidermal osmolyte required to maintain keratinocyte hydration in a dry environment. PMID:12880428

  1. Autocrine growth factors for human tumor clonogenic cells.

    PubMed

    Hamburger, A W; White, C P

    1985-11-01

    A human epithelial-derived cell line, SW-13, releases a soluble substance that functions as an autocrine growth factor. SW-13 cells, derived from a human adenocarcinoma of the adrenal cortex, form a few small colonies when suspended in soft agar at low densities. The number of colonies increased significantly when either viable SW-13 cells or serum-free medium conditioned by SW-13 cells (CM) was added to agar underlayers. CM increased colony formation in a dose-dependent fashion. Clonal growth at low cell densities was dependent on the presence of both horse serum and SW-13 CM. Neither activity alone was capable of sustaining growth. Even when cells were plated at high densities CM could not substitute for serum, but could reduce the threshold serum concentration. The results suggest that autocrine and serum-derived factors act in concert to maintain clonal growth of epithelial tumor cells in soft agar.

  2. Hammerhead Ribozyme-Mediated Knockdown of mRNA for Fibrotic Growth Factors: Transforming Growth Factor-Beta 1 and Connective Tissue Growth Factor

    PubMed Central

    Robinson, Paulette M.; Blalock, Timothy D.; Yuan, Rong; Lewin, Alfred S.; Schultz, Gregory S.

    2013-01-01

    Excessive scarring (fibrosis) is a major cause of pathologies in multiple tissues, including lung, liver, kidney, heart, cornea, and skin. The transforming growth factor- β (TGF- β) system has been shown to play a key role in regulating the formation of scar tissue throughout the body. Furthermore, connective tissue growth factor (CTGF) has been shown to mediate most of the fibrotic actions of TGF- β, including stimulation of synthesis of extracellular matrix and differentiation of fibroblasts into myofibroblasts. Currently, no approved drugs selectively and specifically regulate scar formation. Thus, there is a need for a drug that selectively targets the TGF- β cascade at the molecular level and has minimal off-target side effects. This chapter focuses on the design of hammerhead ribozymes, measurement of kinetic activity, and assessment of knockdown mRNAs of TGF- β and CTGF in cell cultures. PMID:22131029

  3. Stimulatory effect of luteinizing hormone, insulin-like growth factor-1, and epidermal growth factor on vascular endothelial growth factor production in cultured bubaline luteal cells.

    PubMed

    Chouhan, V S; Dangi, S S; Babitha, V; Verma, M R; Bag, S; Singh, G; Sarkar, M

    2015-10-15

    The purpose of this study was to evaluate the temporal (24, 48, and 72 hours) and dose-dependent (0, 5, 10, and 100 ng/mL of LH, insulin-like growth factor 1 [IGF-1], and EGF) in vitro expression and secretion patterns of vascular endothelial growth factor (VEGF) in luteal cell culture during different stages of estrous cycle in water buffaloes. Corpus luteum samples from ovaries of early luteal phase (ELP; Days 1-4), midluteal phase (Days 5-10), and late luteal phase (Days 11-16) were collected from a local slaughterhouse. The samples were then processed and cultured in (serum containing) appropriate cell culture medium and incubated separately with three factors (LH, IGF-1, or EGF) at the previously mentioned three dose-duration combinations. At the end of the respective incubation periods, VEGF was assayed in the spent culture medium by ELISA, whereas the cultured cells were used for VEGF mRNA expression by quantitative real-time polymerase chain reaction. The results of the present study disclosed dose- and time-dependent stimulatory effects of LH, IGF-1, and EGF on VEGF production in bubaline luteal cells. The VEGF expression and secretion from the cultured luteal cells were highest during the ELP, intermediate in the midluteal phase, and lowest in the late luteal phase of the estrous cycle for all the three tested factors. Comparison of the results of the three treatments depicted EGF as the most potent stimulating factor followed by IGF-1 and LH. Immunocytochemistry findings in luteal cell culture of ELP agreed with the VEGF expression and secretion. In conclusion, mRNA expression, protein secretion, and immunolocalization of VEGF data clearly indicated for the first time that LH, IGF-1, and EGF play an important role in stimulating luteal angiogenesis in buffalo CL. The highest expression and secretion of VEGF in the ELP might be associated with the development of blood vessels in early growth of CL, which in turn gets augmented by the aforementioned

  4. Stimulatory effect of luteinizing hormone, insulin-like growth factor-1, and epidermal growth factor on vascular endothelial growth factor production in cultured bubaline luteal cells.

    PubMed

    Chouhan, V S; Dangi, S S; Babitha, V; Verma, M R; Bag, S; Singh, G; Sarkar, M

    2015-10-15

    The purpose of this study was to evaluate the temporal (24, 48, and 72 hours) and dose-dependent (0, 5, 10, and 100 ng/mL of LH, insulin-like growth factor 1 [IGF-1], and EGF) in vitro expression and secretion patterns of vascular endothelial growth factor (VEGF) in luteal cell culture during different stages of estrous cycle in water buffaloes. Corpus luteum samples from ovaries of early luteal phase (ELP; Days 1-4), midluteal phase (Days 5-10), and late luteal phase (Days 11-16) were collected from a local slaughterhouse. The samples were then processed and cultured in (serum containing) appropriate cell culture medium and incubated separately with three factors (LH, IGF-1, or EGF) at the previously mentioned three dose-duration combinations. At the end of the respective incubation periods, VEGF was assayed in the spent culture medium by ELISA, whereas the cultured cells were used for VEGF mRNA expression by quantitative real-time polymerase chain reaction. The results of the present study disclosed dose- and time-dependent stimulatory effects of LH, IGF-1, and EGF on VEGF production in bubaline luteal cells. The VEGF expression and secretion from the cultured luteal cells were highest during the ELP, intermediate in the midluteal phase, and lowest in the late luteal phase of the estrous cycle for all the three tested factors. Comparison of the results of the three treatments depicted EGF as the most potent stimulating factor followed by IGF-1 and LH. Immunocytochemistry findings in luteal cell culture of ELP agreed with the VEGF expression and secretion. In conclusion, mRNA expression, protein secretion, and immunolocalization of VEGF data clearly indicated for the first time that LH, IGF-1, and EGF play an important role in stimulating luteal angiogenesis in buffalo CL. The highest expression and secretion of VEGF in the ELP might be associated with the development of blood vessels in early growth of CL, which in turn gets augmented by the aforementioned

  5. Growth factors with heparin binding affinity in human synovial fluid

    SciTech Connect

    Hamerman, D.; Taylor, S.; Kirschenbaum, I.; Klagsbrun, M.; Raines, E.W.; Ross, R.; Thomas, K.A.

    1987-12-01

    Synovial effusions were obtained from the knees of 15 subjects with joint trauma, menisceal or ligamentous injury, or osteoarthritis. Heparin-Sepharose affinity chromatography of these synovial fluids revealed, in general, three major peaks of mitogenic activity as measured by incorporation of /sup 3/H-thymidine into 3T3 cells. Gradient elution patterns showed activities at 0.5M NaCl, which is characteristic of platelet derived growth factor, and at 1.1 M NaCl and 1.6M NaCl, indicative of acidic and basic fibroblast growth factors, respectively. The identities of these mitogenic fractions were confirmed by specific immunologic and receptor-binding assays. The presence of platelet derived, acidic and basic fibroblast growth factors in the synovial fluid may contribute to wound healing in the arthritic joint.

  6. Epidermal growth factor, from gene organization to bedside

    PubMed Central

    Zeng, Fenghua; Harris, Raymond C.

    2014-01-01

    In 1962, epidermal growth factor (EGF) was discovered by Dr. Stanley Cohen while studying nerve growth factor (NGF). It was soon recognized that EGF is the prototypical member of a family of peptide growth factors that activate the EGF receptors, and that the EGF/EGF receptor signaling pathway plays important roles in proliferation, differentiation and migration of a variety of cell types, especially in epithelial cells. After the basic characterization of EGF function in the first decade or so after its discovery, the studies related to EGF and its signaling pathway have extended to a broad range of investigations concerning its biological and pathophysiological roles in development and in human diseases. In this review, we briefly describe the gene organization and tissue distribution of EGF, with emphasis on its biological and pathological roles in human diseases. PMID:24513230

  7. Keratinocyte stem cells: a commentary.

    PubMed

    Potten, Christopher S; Booth, Catherine

    2002-10-01

    For many years it has been widely accepted that stem cells play a crucial role in adult tissue maintenance. The concept that the renewing tissues of the body contain a small subcompartment of self-maintaining stem cells, upon which the entire tissue is dependent, is also now accepted as applicable to all renewing tissues. Gene therapy and tissue engineering are driving considerable interest in the clinical application of such hierarchically organized cellular compartments. Recent initial observations have provided a tantalizing insight into the large pluripotency of these cells. Indeed, scientists are now beginning to talk about the possible totipotency of some adult tissue stem cells. Such work is currently phenomenologic, but analysis of data derived from genomics and proteomics, identifying the crucial control signals involved, will soon provide a further impetus to stem cell biology with far reaching applications. The epidermis with its relatively simple structure, ease of accessibility, and the ability to grow its cells in vitro is one obvious target tissue for testing stem cell manipulation theories. It is crucial, however, that the normal keratinocyte stem cell is thoroughly characterized prior to attempting to manipulate its pluripotency. This commentary assesses the data generated to date and critically discusses the conclusions that have been drawn. Our current level of understanding, or lack of understanding, of the keratinocyte stem cell is reviewed.

  8. Cultured human foreskin fibroblasts produce a factor that stimulates their growth with properties similar to basic fibroblast growth factor

    SciTech Connect

    Story, M.T. )

    1989-05-01

    To determine if fibroblasts could be a source of fibroblast growth factor (FGF) in tissue, cells were initiated in culture from newborn human foreskin. Fibroblast cell lysates promoted radiolabeled thymidine uptake by cultured quiescent fibroblasts. Seventy-nine percent of the growth-promoting activity of lysates was recovered from heparin-Sepharose. The heparin-binding growth factor reacted on immunoblots with antiserum to human placenta-derived basic FGF and competed with iodinated basic FGF for binding to antiserum to (1-24)bFGF synthetic peptide. To confirm that fibroblasts were the source of the growth factor, cell lysates were prepared from cells incubated with radiolabeled methionine. Heparin affinity purified material was immunoprecipitated with basic FGF antiserum and electrophoresed. Radiolabeled material was detected on gel autoradiographs in the same molecular weight region as authentic iodinated basic FGF. The findings are consistant with the notion that cultured fibroblasts express basic FGF. As these cells also respond to the mitogen, it is possible that the regulation of their growth is under autocrine control. Fibroblasts may be an important source of the growth factor in tissue.

  9. A Model to Predict the Risk of Keratinocyte Carcinomas.

    PubMed

    Whiteman, David C; Thompson, Bridie S; Thrift, Aaron P; Hughes, Maria-Celia; Muranushi, Chiho; Neale, Rachel E; Green, Adele C; Olsen, Catherine M

    2016-06-01

    Basal cell and squamous cell carcinomas of the skin are the commonest cancers in humans, yet no validated tools exist to estimate future risks of developing keratinocyte carcinomas. To develop a prediction tool, we used baseline data from a prospective cohort study (n = 38,726) in Queensland, Australia, and used data linkage to capture all surgically excised keratinocyte carcinomas arising within the cohort. Predictive factors were identified through stepwise logistic regression models. In secondary analyses, we derived separate models within strata of prior skin cancer history, age, and sex. The primary model included terms for 10 items. Factors with the strongest effects were >20 prior skin cancers excised (odds ratio 8.57, 95% confidence interval [95% CI] 6.73-10.91), >50 skin lesions destroyed (odds ratio 3.37, 95% CI 2.85-3.99), age ≥ 70 years (odds ratio 3.47, 95% CI 2.53-4.77), and fair skin color (odds ratio 1.75, 95% CI 1.42-2.15). Discrimination in the validation dataset was high (area under the receiver operator characteristic curve 0.80, 95% CI 0.79-0.81) and the model appeared well calibrated. Among those reporting no prior history of skin cancer, a similar model with 10 factors predicted keratinocyte carcinoma events with reasonable discrimination (area under the receiver operator characteristic curve 0.72, 95% CI 0.70-0.75). Algorithms using self-reported patient data have high accuracy for predicting risks of keratinocyte carcinomas.

  10. Expression and localization of epidermal growth factor, transforming growth factor-α and epidermal growth factor receptor in the canine testis

    PubMed Central

    TAMADA, Hiromichi; TAKEMOTO, Kohei; TOMINAGA, Masato; KAWATE, Noritoshi; TAKAHASHI, Masahiro; HATOYA, Shingo; MATSUYAMA, Satoshi; INABA, Toshio; SAWADA, Tsutomu

    2015-01-01

    Gene expression of epidermal growth factor (EGF), transforming growth factor-α (TGF-α) and EGF receptor (EGF-R) and the localization of the corresponding proteins in the canine testis were studied. Levels of mRNA expressions were determined by semiquantitative reverse transcription polymerase chain reaction in the testes of the peripubertal (4–6 months), young adult (3–4 years), advanced adult (7–8 years) and senescent (11–16 years) groups. The EGF-R mRNA level in the testes of the peripubertal group was significantly higher than those in the other groups, whereas there was no difference in EGF and TGF-α mRNA levels among groups. Immunohistochemical stainings for EGF, TGF-α and EGF-R in the testis revealed that immunoreactivity in the seminiferous epithelium and Sertoli cell was weak and nonspecific for the stage of spermatogenesis, and distinct staining was found in Leydig cells. These results suggest that the EGF family of growth factors may be involved in testicular maturation and function in the dog. PMID:26498203

  11. Effects of growth factors on temporomandibular joint disc cells.

    PubMed

    Detamore, Michael S; Athanasiou, Kyriacos A

    2004-07-01

    The effects of growth factors on cartilaginous tissues are well documented. An exception is the temporomandibular joint (TMJ) disc, where data for growth factor effects on proliferation and biosynthesis are very limited. The purpose of this study was to quantify proliferation of and synthesis by TMJ disc cells cultured in monolayer with either platelet derived growth factor-AB (PDGF), basic fibroblast growth factor (bFGF) or insulin-like growth factor-I (IGF), at either a low (10 ng/ml) or high (100 ng/ml) concentration. Proliferation was assessed with a DNA quantitation technique, collagen synthesis was measured via a hydroxyproline assay, and GAG synthesis was determined with a dimethylmethylene blue dye binding assay at 14 days. Overall, the most beneficial growth factor was bFGF, which was most potent in increasing proliferation and GAG synthesis, and also effective in promoting collagen synthesis. At the high concentration, bFGF resulted in 96% more cells than the control and 30 to 45% more cells than PDGF and IGF. PDGF and bFGF were the most potent upregulators of GAG synthesis, producing 2-3 times more GAG than the control. IGF had no significant effect on GAG production, although at its higher concentration it increased collagen production by 4.5 times over the control. Collagen synthesis was promoted by bFGF at its lower concentration, with levels 4.2 times higher than the control, whereas PDGF had no significant effect on collagen production. In general, higher concentrations increased proliferation, whereas lower concentrations favoured biosynthesis. PMID:15126139

  12. Epidermal Growth Factor-Like Growth Factors in the Follicular Fluid: Role in Oocyte Development and Maturation

    PubMed Central

    Hsieh, Minnie; Zamah, A. Musa; Conti, Marco

    2015-01-01

    The growth and maturation of the ovarian follicle requires the coordinate function of somatic cells and the oocyte. Over the past three decades, numerous growth factors involved in the bidirectional signals between the somatic and germ cells have been identified. A possible function of epidermal growth factor (EGF) signaling at selected stages of follicle maturation had been proposed early on and is supported by many observations of in vitro effects of this growth factor on steroidogenesis, oocyte maturation, and cumulus expansion. However, attempts to link EGF levels in the follicular fluid with the state of follicle and oocyte maturation have been inconclusive. More recently, data generated using mouse genetic models perturbing ovulation and fertility indicate that EGF-like growth factors, rather than EGF itself, accumulate in the follicle at the time of ovulation. EGF-like growth factor mRNA is regulated by the luteinizing hormone surge, and corresponding proteins are detected in the follicle. The EGF-like growth factors amphiregulin, epiregulin, and betacellulin are potent stimulators of oocyte maturation and cumulus expansion, and perturbation of this EGF network in vivo impairs ovulation. Similar findings in species other than the mouse confirm an important physiological role for this network at the time of ovulation. Whether this network also plays a critical role in humans and whether it can be used as a biological marker of follicle development or for the improvement of fertility remains to be determined. This review summarizes the most recent findings on the EGF network during ovulation and the potential clinical applications of manipulating this intercellular communication pathway in the control of fertility. PMID:19197805

  13. Placental Growth Factor Administration Abolishes Placental Ischemia-Induced Hypertension.

    PubMed

    Spradley, Frank T; Tan, Adelene Y; Joo, Woo S; Daniels, Garrett; Kussie, Paul; Karumanchi, S Ananth; Granger, Joey P

    2016-04-01

    Preeclampsia is a pregnancy-specific disorder of new-onset hypertension. Unfortunately, the most effective treatment is early delivery of the fetus and placenta. Placental ischemia appears central to the pathogenesis of preeclampsia because placental ischemia/hypoxia induced in animals by reduced uterine perfusion pressure (RUPP) or in humans stimulates release of hypertensive placental factors into the maternal circulation. The anti-angiogenic factor soluble fms-like tyrosine kinase-1 (sFlt-1), which antagonizes and reduces bioavailable vascular endothelial growth factor and placental growth factor (PlGF), is elevated in RUPP rats and preeclampsia. Although PlGF and vascular endothelial growth factor are both natural ligands for sFlt-1, vascular endothelial growth factor also has high affinity to VEGFR2 (Flk-1) causing side effects like edema. PlGF is specific for sFlt-1. We tested the hypothesis that PlGF treatment reduces placental ischemia-induced hypertension by antagonizing sFlt-1 without adverse consequences to the mother or fetus. On gestational day 14, rats were randomized to 4 groups: normal pregnant or RUPP±infusion of recombinant human PlGF (180 μg/kg per day; AG31, a purified, recombinant human form of PlGF) for 5 days via intraperitoneal osmotic minipumps. On day 19, mean arterial blood pressure and plasma sFlt-1 were higher and glomerular filtration rate lower in RUPP than normal pregnant rats. Infusion of recombinant human PlGF abolished these changes seen with RUPP along with reducing oxidative stress. These data indicate that the increased sFlt-1 and reduced PlGF resulting from placental ischemia contribute to maternal hypertension. Our novel finding that recombinant human PlGF abolishes placental ischemia-induced hypertension, without major adverse consequences, suggests a strong therapeutic potential for this growth factor in preeclampsia. PMID:26831193

  14. Cytokines and growth factors which regulate bone cell function

    NASA Astrophysics Data System (ADS)

    Seino, Yoshiki

    Everybody knows that growth factors are most important in making bone. Hormones enhance bone formation from a long distance. Growth factors promote bone formation as an autocrine or paracrine factor in nearby bone. BMP-2 through BMP-8 are in the TGF-β family. BMP makes bone by enchondral ossification. In bone, IGF-II is most abundant, second, TGF-β, and third IGF-I. TGF-β enhances bone formation mainly by intramembranous ossification in vivo. TGF-β affects both cell proliferation and differentiation, however, TGF-β mainly enhances bone formation by intramembranous ossification. Interestingly, TGF-β is increased by estrogen(E 2), androgen, vitamin D, TGF-β and FGF. IGF-I and IGF-II also enhance bone formation. At present it remains unclear why IGF-I is more active in bone formation than IGF-II, although IGF-II is more abundant in bone compared to IGF-I. However, if only type I receptor signal transduction promotes bone formation, the strong activity of IGF-I in bone formation is understandable. GH, PTH and E 2 promotes IGF-I production. Recent data suggest that hormones containing vitamin D or E 2 enhance bone formation through growth factors. Therefore, growth factors are the key to clarifying the mechanism of bone formation.

  15. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis.

    PubMed Central

    Connolly, D T; Heuvelman, D M; Nelson, R; Olander, J V; Eppley, B L; Delfino, J J; Siegel, N R; Leimgruber, R M; Feder, J

    1989-01-01

    Vascular permeability factor (VPF) is an Mr 40-kD protein that has been purified from the conditioned medium of guinea pig line 10 tumor cells grown in vitro, and increases fluid permeability from blood vessels when injected intradermally. Addition of VPF to cultures of vascular endothelial cells in vitro unexpectedly stimulated cellular proliferation. VPF promoted the growth of new blood vessels when administered into healing rabbit bone grafts or rat corneas. The identity of the growth factor activity with VPF was established in four ways: (a) the molecular weight of the activity in preparative SDS-PAGE was the same as VPF (Mr approximately 40 kD); (b) multiple isoforms (pI greater than or equal to 8) for both VPF and the growth-promoting activity were observed; (c) a single, unique NH2-terminal amino acid sequence was obtained; (d) both growth factor and permeability-enhancing activities were immunoadsorbed using antipeptide IgG that recognized the amino terminus of VPF. Furthermore, 125I-VPF was shown to bind specifically and with high affinity to endothelial cells in vitro and could be chemically cross-linked to a high-molecular weight cell surface receptor, thus demonstrating a mechanism whereby VPF can interact directly with endothelial cells. Unlike other endothelial cell growth factors, VPF did not stimulate [3H]thymidine incorporation or promote growth of other cell types including mouse 3T3 fibroblasts or bovine smooth muscle cells. VPF, therefore, appears to be unique in its ability to specifically promote increased vascular permeability, endothelial cell growth, and angio-genesis. Images PMID:2478587

  16. Differential response of human adipose tissue-derived mesenchymal stem cells, dermal fibroblasts, and keratinocytes to burn wound exudates: potential role of skin-specific chemokine CCL27.

    PubMed

    van den Broek, Lenie J; Kroeze, Kim L; Waaijman, Taco; Breetveld, Melanie; Sampat-Sardjoepersad, Shakun C; Niessen, Frank B; Middelkoop, Esther; Scheper, Rik J; Gibbs, Susan

    2014-01-01

    Many cell-based regenerative medicine strategies toward tissue-engineered constructs are currently being explored. Cell-cell interactions and interactions with different biomaterials are extensively investigated, whereas very few studies address how cultured cells will interact with soluble wound-healing mediators that are present within the wound bed after transplantation. The aim of this study was to determine how adipose tissue-derived mesenchymal stem cells (ASC), dermal fibroblasts, and keratinocytes will react when they come in contact with the deep cutaneous burn wound bed. Burn wound exudates isolated from deep burn wounds were found to contain many cytokines, including chemokines and growth factors related to inflammation and wound healing. Seventeen mediators were identified by ELISA (concentration range 0.0006-9 ng/mg total protein), including the skin-specific chemokine CCL27. Burn wound exudates activated both ASC and dermal fibroblasts, but not keratinocytes, to increase secretion of CXCL1, CXCL8, CCL2, and CCL20. Notably, ASC but not fibroblasts or keratinocytes showed significant increased secretion of vascular endothelial growth factor (5-fold) and interleukin-6 (253-fold), although when the cells were incorporated in bi-layered skin substitute (SS) these differences were less pronounced. A similar discrepancy between ASC and dermal fibroblast mono-cultures was observed when recombinant human-CCL27 was used instead of burn wound exudates. Although CCL27 did not stimulate the secretion of any of the wound-healing mediators by keratinocytes, these cells, in contrast to ASC or dermal fibroblasts, showed increased proliferation and migration. Taken together, these results indicate that on transplantation, keratinocytes are primarily activated to promote wound closure. In contrast, dermal fibroblasts and, in particular, ASC respond vigorously to factors present in the wound bed, leading to increased secretion of angiogenesis/granulation tissue formation

  17. Vascular endothelial growth factors: A comparison between invertebrates and vertebrates.

    PubMed

    Kipryushina, Yulia O; Yakovlev, Konstantin V; Odintsova, Nelly A

    2015-12-01

    This review aims to summarize recent data concerning the structure and role of the members of the vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor (VEGFR) families in the context of early development, organogenesis and regeneration, with a particular emphasis on the role of these factors in the development of invertebrates. Homologs of VEGF and/or VEGFR have been found in all Eumetazoa, in both Radiata and Bilateria, where they are expressed in the descendants of different germ layers and play a pivotal role in the development of animals with and without a vascular system. VEGF is a well-known angiogenesis regulator, but this factor also control cell migration during neurogenesis and the development of branching organs (the trachea) in invertebrate and vertebrate species. A possible explanation for the origin of Vegf/Vegfr in the animal kingdom and a pathway of Vegf/Vegfr evolution are discussed.

  18. Growth factors in the treatment of early osteoarthritis

    PubMed Central

    Civinini, Roberto; Nistri, Lorenzo; Martini, Caterina; Redl, Birgit; Ristori, Gabriele; Innocenti, Massimo

    2013-01-01

    Summary Regenerative medicine is the science that studies the regeneration of biological tissues obtained through use of cells, with the aid of support structures and with biomolecules such as growth factors. As regards the growth factors the PRP, or the platelet-rich plasma, obtained from a withdrawal of autologous blood, concentrating the platelets, represents a safe, economical, easy to prepare and easy to apply source of growth factors. Numerous growth factors are in fact within the platelets and in particular a large number of them have a specific activity on neo-proliferation, on cartilage regeneration and in particular also an antiapoptotic effect on chondroblasts: - The PDGF which regulates the secretion and synthesis of collagen;- The EGF that causes cellular proliferation, endothelial chemotaxis and angiogenesis;- The VEGF that increases angiogenesis and vascular permeability;- The TGF-beta that stimulates the proliferation of undifferentiated MSC, stimulates chemotaxis of endothelial cells and angiogenesis;- The bFGF that promotes the growth and differentiation of chondrocytes and osteoblasts stimulates mitogenesis of mesenchymal cells, chondrocytes and osteoblasts. These properties have led to the development of studies that evaluated the efficacy of treatment of infiltrations in the knee and hip with platelet-derived growth factors. Regarding the knee it was demonstrated that in patients with moderate degree of gonarthrosis, the PRP is able to significantly reduce the pain and improve joint function, both on placebo and towards infiltrations with hyaluronic acid. The success of the treatment was proportional to the age of and inversely proportional to the severity of osteoarthritis according to Kellgren and Lawrence classification. The possibility of infiltrations guided with ultrasound into the hip led us to extend the indications also to hip arthrosis, as already showed by Sanchez. Even in coxarthrosis preliminary results at 6 and 12 months show that

  19. Factors affecting Staphylococcus epidermidis growth in peritoneal dialysis solutions.

    PubMed Central

    McDonald, W A; Watts, J; Bowmer, M I

    1986-01-01

    Staphylococcus epidermidis is the most frequent cause of peritonitis complicating continuous ambulatory peritoneal dialysis. We studied factors that might influence the growth of S. epidermidis in commercially available peritoneal dialysis solution (PDS). Test strains were inoculated into PDS and incubated overnight at 37 degrees C. Samples were removed at appropriate intervals, bacterial counts were performed, and growth curves were constructed. We studied the effects of various osmolarities, the neutralization and acidification of fresh and spent PDS, and the effect of intraperitoneal dwell time on the ability PDS to support growth of S. epidermidis. In fresh PDS, numbers of bacteria remained constant after 24 h. No significant differences in growth were observed among PDS with 0.5, 1.5, 2.5, and 4.25% glucose. Neutralizing acidic fresh PDS had no effect on bacterial growth. However, growth did occur in spent PDS. PDS which was recovered after only 2 h in the peritoneal cavity supported growth to the same extent as did PDS recovered after 4 to 6 h. Mean log10 changes after 24 h of incubation were as follows: for fresh PDS, -1.3; after 2 h dwell time, 2.9; after 4 h dwell time, 1.9; and after 6 h dwell time, 1.3. Acidification of spent PDS to less than pH 6.35 produced less rapid growth; mean log10 increases after 24 h of incubation were 1.9 for pH 7.75, 1.6 for pH 6.35, 0.6 for pH 5.75, and 0.7 for pH 4.95. Fresh PDS of all available osmolarities neither supported the growth of S. epidermidis nor was bactericidal. Spent PDS supported bacterial growth, and this growth was partly independent of the neutralization which occurred during the dialysis. PMID:3722356

  20. Effects of epidermal Langerhans cell's conditioned medium on keratinocytes: a role of Langerhans cells in cholesteatoma.

    PubMed

    Kamide, Y; Sasaki, H; Abramson, M; Huang, C C

    1991-01-01

    Langerhans cells (LCs) are known to play an important role in the immunosurveillance system. In this study, as in others, numerous LCs were detected in the epithelial layer of acquired cholesteatoma by immunohistochemical staining. This finding suggests that cell-mediated immune responses are initiated by LCs in cholesteatoma; however, documentation concerning the microenvironment of LCs-keratinocytes in cholesteatoma is limited. Therefore, we investigated the effects of LCs on keratinocytes in vitro. To study these effects it was necessary to isolate and purify LCs. Our present study revealed that good enrichment and a high degree of purity (95%) of LCs could be obtained from neonatal rat skin using the immunomagnetic beads (Dynabeads M-450) sorting technique. These isolated LCs have the biologic activity of LCs, and Langerhans cells' conditioned medium (LCCM) stimulates DNA synthesis in thymocytes. The effect of LCCM on keratinocytes was then studied. We found that (1) LCCM stimulated DNA synthesis in keratinocytes was then studied. We found that (1) LCCM stimulated DNA synthesis in keratinocytes, but not protein synthesis, and (2) LCCM stimulated the incorporation of 3H-putrescine into keratinocytes by the activation of transglutaminase. Transglutaminase is a known marker of terminal differentiation in keratinocytes. By Western blot analysis, we identified a 17-kd immunoreactive mouse interleukin-1 alpha in LCCM. Our results imply that LCs found in cholesteatoma tissue may play an important role in stimulating both hyper-proliferation and cornification of keratinocytes; two characteristic features of cholesteatoma formation. These stimulatory effects may be due to the release of interleukin-1 or other factors by LCs.

  1. Binding, sequestration, and processing of epidermal growth factor and nerve growth factor by PC12 cells. [Rats

    SciTech Connect

    Chandler, C.E.; Herschman, H.R.

    1983-03-01

    Th rat PC12 pheochromocytoma cell line exhibits biological responses to both nerve growth factor (NGF) and epidermal growth factor (EGF). The existence of receptors and biological responses on a common cell for these two well-characterized polypeptide growth factors makes this an attractive system for comparison of ligand binding and processing. Both NGF and EGF are bound to PC12 cells in a competable form at 4/sup 0/C. At 37/sup 0/C both ligands are ''sequestered,'' but at different rates and to different extents. While sequestration happens rapidly and nearly quantitatively for bound EGF, the dissociation reaction appears to compete favorably with NFG sequestration. Both EGF and NGF are degraded by PC12 cells. Sequestered EGF, however, is degraded to a greater extent than sequestered NGF.

  2. In vitro investigations on the effect of dermal fibroblasts on keratinocyte responses to ultraviolet B radiation.

    PubMed

    Fernandez, Tara L; Van Lonkhuyzen, Derek R; Dawson, Rebecca A; Kimlin, Michael G; Upton, Zee

    2014-01-01

    Exposure to ultraviolet radiation is closely linked to the development of skin cancers in humans. The ultraviolet B (UVB) radiation wavelength (280-320 nm), in particular, causes DNA damage in epidermal keratinocytes, which are linked to the generation of signature premalignant mutations. Interactions between dermal fibroblasts and keratinocytes play a role in epidermal repair and regeneration after UVB-induced damage. To investigate these processes, established two and three-dimensional culture models were utilized to study the impact of fibroblast-keratinocyte crosstalk during the acute UVB response. Using a coculture system it was observed that fibroblasts enhanced keratinocyte survival and the repair of cyclobutane pyrimidine dimers (CPDs) after UVB radiation exposure. These findings were also mirrored in irradiated human skin coculture models employed in this study. Fibroblast coculture was shown to play a role in the expression and activation of members of the apoptotic cascade, including caspase-3 and Bad. Interestingly, the expression and phosphorylation of p53, a key player in the regulation of keratinocyte cell fate postirradiation, was also shown to be influenced by fibroblast-produced factors. This study highlights the importance of synergistic interactions between fibroblasts and keratinocytes in maintaining a functional epidermis while promoting repair and regeneration following UVB radiation-induced damage.

  3. Endothelin-1 of keratinocyte origin is a mediator of melanocyte dendricity.

    PubMed

    Hara, M; Yaar, M; Gilchrest, B A

    1995-12-01

    Melanocytes synthesize melanin and transfer it to keratinocytes via dendritic processes. Keratinocytes are known to produce constitutively several factors, including endothelin-1 (ET-1), that together affect melanocyte proliferation, migration, melanogenesis, and dendrite formation. After ultraviolet (UV) irradiation, synthesis and secretion of ET-1 are up-regulated in keratinocytes. Because UV irradiation of skin is known to be associated with increased melanocyte dendricity, and because medium conditioned by UV-irradiated keratinocytes (UV-KCM) induces melanocyte dendricity to a greater degree than does baseline keratinocyte-conditioned medium (KCM), we investigated whether ET-1 promotes melanocyte dendricity. ET-1, originally recognized as a vasoconstrictive peptide, has recently been shown to stimulate melanocyte proliferation and tyrosinase activity. We now report that ET-1 supplementation of cultured melanocytes significantly increases the percentage of dendritic melanocytes, as well as dendrite length, in a dose-dependent manner. Moreover, UV-KCM was found to contain over 25-fold more ET-1 than KCM, and ET-1 supplementation of KCM induced melanocyte dendricity comparable to that induced by UV-KCM. Further, melanocyte dendricity induced by UV-KCM was significantly inhibited by the addition of anti-ET-1 monoclonal antibody to the medium, suggesting that the UV-KCM effect on melanocyte dendricity is mediated largely through ET-1. Our findings suggest that in the skin, ET-1 of keratinocyte origin promotes melanocyte dendricity in response to UV irradiation.

  4. Differentiation of Keratinocytes Modulates Skin HPA Analog.

    PubMed

    Wierzbicka, Justyna M; Żmijewski, Michał A; Antoniewicz, Jakub; Sobjanek, Michal; Slominski, Andrzej T

    2017-01-01

    It is well established, that epidermal keratinocytes express functional equivalent of hypothalamus-pituitary-adrenal axis (HPA) in order to respond to changing environment and maintain internal homeostasis. We are presenting data indicating that differentiation of primary neonatal human keratinocytes (HPEKp), induced by prolonged incubation or calcium is accompanied by significant changes in the expression of the elements of skin analog of HPA (sHPA). Expression of CRF, UCN1-3, POMC, ACTH, CRFR1, CRFR2, MC1R, MC2R, and GR (coded by NR3C1 gene) were observed on gene/protein levels along differentiation of keratinocytes in culture with similar pattern seen by immunohistochemistry on full thickness skin biopsies. Expression of CRF was more pronounced in less differentiated keratinocytes, which corresponded to the detection of CRF immunoreactivity preferentially in the stratum basale. POMC expression was enhanced in more differentiated keratinocytes, which corresponded to detection of ACTH immunoreactivity, predominantly in the stratum spinosum and stratum granulosum. Expression of urocortins was also affected by induction of HPEKp differentiation. Immunohistochemical studies showed high prevalence of CRFR1 in well differentiated keratinocytes, while smaller keratinocytes showed predominantly CRFR2 immunoreactivity. MC2R mRNA levels were elevated from days 4 to 8 of in vitro incubation, while MC2R immunoreactivity was the highest in the upper layers of epidermis. Similar changes in mRNA/protein levels of sHPA elements were observed in HPEKp keratinocytes treated with calcium. Summarizing, preferential expression of CRF and POMC (ACTH) by populations of keratinocytes on different stage of differentiation resembles organization of central HPA axis suggesting their distinct role in physiology and pathology of the epidermis. J. Cell. Physiol. 232: 154-166, 2017. © 2016 Wiley Periodicals, Inc.

  5. Single cell mechanics of keratinocyte cells.

    PubMed

    Lulevich, Valentin; Yang, Hsin-ya; Isseroff, R Rivkah; Liu, Gang-yu

    2010-11-01

    Keratinocytes represent the major cell type of the uppermost layer of human skin, the epidermis. Using AFM-based single cell compression, the ability of individual keratinocytes to resist external pressure and global rupturing forces is investigated and compared with various cell types. Keratinocytes are found to be 6-70 times stiffer than other cell types, such as white blood, breast epithelial, fibroblast, or neuronal cells, and in contrast to other cell types they retain high mechanic strength even after the cell's death. The absence of membrane rupturing peaks in the force-deformation profiles of keratinocytes and their high stiffness during a second load cycle suggests that their unique mechanical resistance is dictated by the cytoskeleton. A simple analytical model enables the quantification of Young's modulus of keratinocyte cytoskeleton, as high as 120-340 Pa. Selective disruption of the two major cytoskeletal networks, actin filaments and microtubules, does not significantly affect keratinocyte mechanics. F-actin is found to impact cell deformation under pressure. During keratinocyte compression, the plasma membrane stretches to form peripheral blebs. Instead of blebbing, cells with depolymerized F-actin respond to pressure by detaching the plasma membrane from the cytoskeleton underneath. On the other hand, the compression force of keratinocytes expressing a mutated keratin (cell line, KEB-7) is 1.6-2.2 times less than that for the control cell line that has normal keratin networks. Therefore, we infer that the keratin intermediate filament network is responsible for the extremely high keratinocyte stiffness and resilience. This could manifest into the rugged protective nature of the human epidermis. PMID:20728993

  6. Prodomains of transforming growth factor beta (TGFbeta) superfamily members specify different functions: extracellular matrix interactions and growth factor bioavailability.

    PubMed

    Sengle, Gerhard; Ono, Robert N; Sasaki, Takako; Sakai, Lynn Y

    2011-02-18

    The specific functions of the prodomains of TGFβ superfamily members are largely unknown. Interactions are known between prodomains of TGFβ-1-3 and latent TGFβ-binding proteins and between prodomains of BMP-2, -4, -7, and -10 and GDF-5 and fibrillins, raising the possibility that latent TGFβ-binding proteins and fibrillins may mediate interactions with all other prodomains of this superfamily. This possibility is tested in this study. Results show that the prodomain of BMP-5 interacts with the N-terminal regions of fibrillin-1 and -2 in a site similar to the binding sites for other bone morphogenetic proteins. However, in contrast, the prodomain of GDF-8 (myostatin) interacts with the glycosaminoglycan side chains of perlecan. The binding site for the GDF-8 prodomain is likely the heparan sulfate chain present on perlecan domain V. These results support and extend the emerging concept that TGFβ superfamily prodomains target their growth factor dimers to extracellular matrix macromolecules. In addition, biochemical studies of prodomain·growth factor complexes were performed to identify inactive complexes. For some members of the superfamily, the prodomain is noncovalently associated with its growth factor dimer in an inactive complex; for others, the prodomain·growth factor complex is active, even though the prodomain is noncovalently associated with its growth factor dimer. Results show that the BMP-10 prodomain, in contrast to BMP-4, -5, and -7 prodomains, can inhibit the bioactivity of the BMP-10 growth factor and suggest that the BMP-10 complex is like TGFβ and GDF-8 complexes, which can be activated by cleavage of the associated prodomain.

  7. Lactobacillus rhamnosus GG inhibits the toxic effects of Staphylococcus aureus on epidermal keratinocytes.

    PubMed

    Mohammedsaeed, Walaa; McBain, Andrew J; Cruickshank, Sheena M; O'Neill, Catherine A

    2014-09-01

    Few studies have evaluated the potential benefits of the topical application of probiotic bacteria or material derived from them. We have investigated whether a probiotic bacterium, Lactobacillus rhamnosus GG, can inhibit Staphylococcus aureus infection of human primary keratinocytes in culture. When primary human keratinocytes were exposed to S. aureus, only 25% of the keratinocytes remained viable following 24 h of incubation. However, in the presence of 10(8) CFU/ml of live L. rhamnosus GG, the viability of the infected keratinocytes increased to 57% (P = 0.01). L. rhamnosus GG lysates and spent culture fluid also provided significant protection to keratinocytes, with 65% (P = 0.006) and 57% (P = 0.01) of cells, respectively, being viable following 24 h of incubation. Keratinocyte survival was significantly enhanced regardless of whether the probiotic was applied in the viable form or as cell lysates 2 h before or simultaneously with (P = 0.005) or 12 h after (P = 0.01) S. aureus infection. However, spent culture fluid was protective only if added before or simultaneously with S. aureus. With respect to mechanism, both L. rhamnosus GG lysate and spent culture fluid apparently inhibited adherence of S. aureus to keratinocytes by competitive exclusion, but only viable bacteria or the lysate could displace S. aureus (P = 0.04 and 0.01, respectively). Furthermore, growth of S. aureus was inhibited by either live bacteria or lysate but not spent culture fluid. Together, these data suggest at least two separate activities involved in the protective effects of L. rhamnosus GG against S. aureus, growth inhibition and reduction of bacterial adhesion.

  8. Functional upregulation of system xc- by fibroblast growth factor-2.

    PubMed

    Liu, Xiaoqian; Resch, Jon; Rush, Travis; Lobner, Doug

    2012-02-01

    The cystine/glutamate antiporter (system xc-) is a Na(+)-independent amino acid transport system. Disruption of this system may lead to multiple effects in the CNS including decreased cellular glutathione. Since multiple neurological diseases involve glutathione depletion, and disruption of growth factor signaling has also been implicated in these diseases, it is possible that some growth factors effects are mediated by regulation of system xc-. We tested the growth factors fibroblast growth factor-2 (FGF-2), insulin-like growth factor-1 (IGF-1), neuregulin-1 (NRG), neurotrophin-4 (NT-4), and brain derived neurotrophic factor (BDNF) on system xc- mediated 14C-cystine uptake in mixed neuronal and glial cortical cultures. Only FGF-2 significantly increased cystine uptake. The effect was observed in astrocyte-enriched cultures, but not in cultures of neurons or microglia. The increase was blocked by the system xc- inhibitor (s)-4-carboxyphenylglycine, required at least 12 h FGF-2 treatment, and was prevented by the protein synthesis inhibitor cycloheximide. Kinetic analysis indicated FGF-2 treatment increased the V(max) for cystine uptake while the K(m) remained the same. Quantitative PCR showed an increase in mRNA for xCT, the functional subunit of system xc-, beginning at 3 h of FGF-2 treatment, with a dramatic increase after 12 h. Blocking FGFR1 with PD 166866 blocked the FGF-2 effect. Treatment with a PI3-kinase inhibitor (LY-294002) or a MEK/ERK inhibitor (U0126) for 1 h prior to and during the FGF-2 treatment, each partially blocked the increased cystine uptake. The upregulation of system xc- by FGF-2 may be responsible for some of the known physiological actions of FGF-2. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

  9. Priming Dental Pulp Stem Cells With Fibroblast Growth Factor-2 Increases Angiogenesis of Implanted Tissue-Engineered Constructs Through Hepatocyte Growth Factor and Vascular Endothelial Growth Factor Secretion.

    PubMed

    Gorin, Caroline; Rochefort, Gael Y; Bascetin, Rumeyza; Ying, Hanru; Lesieur, Julie; Sadoine, Jérémy; Beckouche, Nathan; Berndt, Sarah; Novais, Anita; Lesage, Matthieu; Hosten, Benoit; Vercellino, Laetitia; Merlet, Pascal; Le-Denmat, Dominique; Marchiol, Carmen; Letourneur, Didier; Nicoletti, Antonino; Vital, Sibylle Opsahl; Poliard, Anne; Salmon, Benjamin; Muller, Laurent; Chaussain, Catherine; Germain, Stéphane

    2016-03-01

    Tissue engineering strategies based on implanting cellularized biomaterials are promising therapeutic approaches for the reconstruction of large tissue defects. A major hurdle for the reliable establishment of such therapeutic approaches is the lack of rapid blood perfusion of the tissue construct to provide oxygen and nutrients. Numerous sources of mesenchymal stem cells (MSCs) displaying angiogenic potential have been characterized in the past years, including the adult dental pulp. Establishment of efficient strategies for improving angiogenesis in tissue constructs is nevertheless still an important challenge. Hypoxia was proposed as a priming treatment owing to its capacity to enhance the angiogenic potential of stem cells through vascular endothelial growth factor (VEGF) release. The present study aimed to characterize additional key factors regulating the angiogenic capacity of such MSCs, namely, dental pulp stem cells derived from deciduous teeth (SHED). We identified fibroblast growth factor-2 (FGF-2) as a potent inducer of the release of VEGF and hepatocyte growth factor (HGF) by SHED. We found that FGF-2 limited hypoxia-induced downregulation of HGF release. Using three-dimensional culture models of angiogenesis, we demonstrated that VEGF and HGF were both responsible for the high angiogenic potential of SHED through direct targeting of endothelial cells. In addition, FGF-2 treatment increased the fraction of Stro-1+/CD146+ progenitor cells. We then applied in vitro FGF-2 priming to SHED before encapsulation in hydrogels and in vivo subcutaneous implantation. Our results showed that FGF-2 priming is more efficient than hypoxia at increasing SHED-induced vascularization compared with nonprimed controls. Altogether, these data demonstrate that FGF-2 priming enhances the angiogenic potential of SHED through the secretion of both HGF and VEGF.

  10. Therapeutic potential of growth factors and their antagonists.

    PubMed Central

    Garner, A.

    1992-01-01

    This article describes studies with four peptides, epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), gastrin-releasing peptide/bombesin (GRP), and gastrin. The mitogenic and anti-secretory activities of EGF/TGF alpha appear to be mediated by a single class of high-affinity membrane receptors but may involve different signal transducing mechanisms. Biological activity of EGF resides in the N-terminal 42 amino acid fragment with the C-terminal undecapeptide determining binding affinity. A parenteral depot formulation of an EGF-related peptide or a small molecule agonist of the EGF receptor could have utility in treating various ulcerative disorders of the gut. Although antagonism of EGF (and thus TGF alpha) receptors and/or transducing mechanisms is frequently cited as a potential therapeutic approach to hyperproliferative diseases, blocking the action of TGF alpha, GRP, or gastrin with neutralizing antibodies or receptor antagonists did not influence the growth of a wide range of solid tumors in nude mice. These findings suggest that, unless tumor growth displays absolute dependency on one particular mitogen, antagonism of a specific growth factor is unlikely to have great effect in cancer therapy. PMID:1341074

  11. Assessing the Factors of Regional Growth Decline of Sugar Maple

    NASA Astrophysics Data System (ADS)

    Bishop, D. A.; Beier, C. M.; Pederson, N.; Lawrence, G. B.; Stella, J. C.; Sullivan, T. J.

    2014-12-01

    Sugar maple (Acer saccharum Marsh) is among the most ecologically, economically and culturally important trees in North America, but has experienced a decline disease across much of its range. We investigated the climatic and edaphic factors associated with A. saccharum growth in the Adirondack Mountains (USA) using a well-replicated tree-ring network incorporating a range of soil fertility (base cation availability). We found that nearly 3 in 4 A. saccharum trees exhibited declining growth rates during the last several decades, regardless of tree age or size. Although diameter growth was consistently higher on base-rich soils, the negative trends in growth were largely consistent across the soil chemistry gradient. Sensitivity of sugar maple growth to climatic variability was overall weaker than expected, but were also non-stationary during the 20th century. We observed increasingly positive responses to late-winter precipitation, increasingly negative responses to growing season temperatures, and strong positive responses to moisture availability during the 1960s drought that became much weaker during the recent pluvial. Further study is needed of these factors and their interactions as potential mechanisms for sugar maple growth decline.

  12. Exosomes released by keratinocytes modulate melanocyte pigmentation.

    PubMed

    Lo Cicero, Alessandra; Delevoye, Cédric; Gilles-Marsens, Floriane; Loew, Damarys; Dingli, Florent; Guéré, Christelle; André, Nathalie; Vié, Katell; van Niel, Guillaume; Raposo, Graça

    2015-01-01

    Cells secrete extracellular vesicles (EVs), exosomes and microvesicles, which transfer proteins, lipids and RNAs to regulate recipient cell functions. Skin pigmentation relies on a tight dialogue between keratinocytes and melanocytes in the epidermis. Here we report that exosomes secreted by keratinocytes enhance melanin synthesis by increasing both the expression and activity of melanosomal proteins. Furthermore, we show that the function of keratinocyte-derived exosomes is phototype-dependent and is modulated by ultraviolet B. In sum, this study uncovers an important physiological function for exosomes in human pigmentation and opens new avenues in our understanding of how pigmentation is regulated by intercellular communication in both healthy and diseased states.

  13. Assaying binding of nerve growth factor to cell surface receptors

    SciTech Connect

    Vale, R.D.; Shooter, E.M.

    1985-01-01

    The paper describes methods both for the radioiodination of nerve growth factor (NGF) and for assaying NFG receptors by reversible binding techniques. Preparation of (/sup 125/I)NGF along with a rapid method for determining the amount of cell-bound ligand have allowed the detection of NGF receptors on a number of cell types.

  14. Role of fibroblast growth factors in organ regeneration and repair.

    PubMed

    El Agha, Elie; Kosanovic, Djuro; Schermuly, Ralph T; Bellusci, Saverio

    2016-05-01

    In its broad sense, regeneration refers to the renewal of lost cells, tissues or organs as part of the normal life cycle (skin, hair, endometrium etc.) or as part of an adaptive mechanism that organisms have developed throughout evolution. For example, worms, starfish and amphibians have developed remarkable regenerative capabilities allowing them to voluntarily shed body parts, in a process called autotomy, only to replace the lost parts afterwards. The bizarre myth of the fireproof homicidal salamander that can survive fire and poison apple trees has persisted until the 20th century. Salamanders possess one of the most robust regenerative machineries in vertebrates and attempting to draw lessons from limb regeneration in these animals and extrapolate the knowledge to mammals is a never-ending endeavor. Fibroblast growth factors are potent morphogens and mitogens that are highly conserved among the animal kingdom. These growth factors play key roles in organogenesis during embryonic development as well as homeostatic balance during postnatal life. In this review, we provide a summary about the current knowledge regarding the involvement of fibroblast growth factor signaling in organ regeneration and repair. We also shed light on the use of these growth factors in previous and current clinical trials in a wide array of human diseases.

  15. Controlled growth factor release from synthetic extracellular matrices

    NASA Astrophysics Data System (ADS)

    Lee, Kuen Yong; Peters, Martin C.; Anderson, Kenneth W.; Mooney, David J.

    2000-12-01

    Polymeric matrices can be used to grow new tissues and organs, and the delivery of growth factors from these matrices is one method to regenerate tissues. A problem with engineering tissues that exist in a mechanically dynamic environment, such as bone, muscle and blood vessels, is that most drug delivery systems have been designed to operate under static conditions. We thought that polymeric matrices, which release growth factors in response to mechanical signals, might provide a new approach to guide tissue formation in mechanically stressed environments. Critical design features for this type of system include the ability to undergo repeated deformation, and a reversible binding of the protein growth factors to polymeric matrices to allow for responses to repeated stimuli. Here we report a model delivery system that can respond to mechanical signalling and upregulate the release of a growth factor to promote blood vessel formation. This approach may find a number of applications, including regeneration and engineering of new tissues and more general drug-delivery applications.

  16. Immunocytochemical expression of growth factors by odontogenic jaw cysts.

    PubMed Central

    Li, T.; Browne, R. M.; Matthews, J. B.

    1997-01-01

    AIM: To determine the immunocytochemical pattern of expression of transforming growth factor (TGF) alpha, epidermal growth factor (EGF), and TGF beta in the three most common types of odontogenic jaw cyst. METHODS: Growth factor expression was detected in paraffin wax sections of odontogenic cysts (27 odontogenic keratocysts, 10 dentigerous cysts, and 10 radicular cysts) using a streptavidin-biotin peroxidase technique with monoclonal antibodies directed against TGF alpha (clone 213-4.4) and TGF beta (clone TB21) and a polyclonal antibody directed against EGF (Z-12). RESULTS: The epithelial linings of all cysts showed reactivity for TGF alpha which was mainly localised to basal and suprabasal layers. Odontogenic keratocyst linings expressed higher levels of TGF alpha than those of dentigerous and radicular cysts, with 89% (24/27) of odontogenic keratocysts exhibiting a strong positive reaction compared with 50% (five of 10) of dentigerous and radicular cysts, respectively. EGF reactivity was similar in all cyst groups, weaker than that for TGF alpha and predominantly suprabasal. TGF alpha and EGF were also detected in endothelial cells, fibroblasts and inflammatory cells within the cyst walls. The most intense TGF beta staining in odontogenic cysts was extracellular within the fibrous tissue capsules, irrespective of cyst type. CONCLUSIONS: These results, together with previous studies of EGF receptor, indicate differential expression of TGF alpha, EGF and their common receptor between the different types of odontogenic cyst, suggesting that these growth factors (via autocrine or paracrine, or both, pathways) may be involved in their pathogenesis. Images PMID:9208810

  17. Fibroblast Growth Factor-2 Alters the Nature of Extinction

    ERIC Educational Resources Information Center

    Graham, Bronwyn M.; Richardson, Rick

    2011-01-01

    These experiments examined the effects of the NMDA-receptor (NMDAr) antagonist MK801 on reacquisition and re-extinction of a conditioned fear that had been previously extinguished before injection of fibroblast growth factor-2 (FGF2) or vehicle. Recent findings have shown that relearning and re-extinction, unlike initial learning and extinction,…

  18. Total Chemical Synthesis of Biologically Active Vascular Endothelial Growth Factor

    SciTech Connect

    Mandal, Kalyaneswar; Kent, Stephen B.H.

    2011-09-15

    The 204-residue covalent-dimer vascular endothelial growth factor (VEGF, see picture) with full mitogenic activity was prepared from three unprotected peptide segments by one-pot native chemical ligations. The covalent structure of the synthetic VEGF was confirmed by precise mass measurement, and the three-dimensional structure of the synthetic protein was determined by high-resolution X-ray crystallography.

  19. A fully autologous co-culture system utilising non-irradiated autologous fibroblasts to support the expansion of human keratinocytes for clinical use.

    PubMed

    Jubin, K; Martin, Y; Lawrence-Watt, D J; Sharpe, J R

    2011-12-01

    Autologous keratinocytes can be used to augment cutaneous repair, such as in the treatment of severe burns and recalcitrant ulcers. Such cells can be delivered to the wound bed either as a confluent sheet of cells or in single-cell suspension. The standard method for expanding primary human keratinocytes in culture uses lethally irradiated mouse 3T3 fibroblasts as feeder cells to support keratinocyte attachment and growth. In an effort to eliminate xenobiotic cells from clinical culture protocols where keratinocytes are applied to patients, we investigated whether human autologous primary fibroblasts could be used to expand keratinocytes in culture. At a defined ratio of a 6:1 excess of keratinocytes to fibroblasts, this co-culture method displayed a population doubling rate comparable to culture with lethally irradiated 3T3 cells. Furthermore, morphological and molecular analysis showed that human keratinocytes expanded in co-culture with autologous human fibroblasts were positive for proliferation markers and negative for differentiation markers. Keratinocytes expanded by this method thus retain their proliferative phenotype, an important feature in enhancing rapid wound closure. We suggest that this novel co-culture method is therefore suitable for clinical use as it dispenses with the need for lethally irradiated 3T3 cells in the rapid expansion of autologous human keratinocytes.

  20. Megakaryocyte growth and development factor is a potent growth factor for primitive hematopoietic progenitors in the human fetus.

    PubMed

    Muench, Marcus O; Bárcena, Alicia

    2004-06-01

    Megakaryocyte growth and development factor (MGDF), or thrombopoietin, has received considerable attention as a therapeutic agent for treating thrombocytopenia or for its use in the ex vivo culture of hematopoietic stem cells. MGDF is known to support the growth of a broad spectrum of hematopoietic precursors obtained from adult or neonatal tissues, but its effects on the growth of fetal progenitors and stem cells has not been studied. Human CD38(+)CD34(2+) progenitors and CD38(-)CD34(2+) cells, a population that contains stem cells, were isolated from midgestation liver and grown under defined conditions with MGDF and various cytokines known to support the growth of primitive hematopoietic precursors. In clonal assays of colony-forming cells (CFCs), MGDF supported the growth of 15-25% of candidate stem cells when combined with granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), flk-2/flt3 ligand, or stem cell factor. MGDF was observed to strongly support the early stages of hematopoiesis and expansion of high proliferative potential CFCs. More mature progenitors were expanded nearly 78-fold in 1 wk of culture with MGDF+SCF+GM-CSF. MGDF alone was also found to support the short-term (2 d) survival of CD38(-)CD34(2+) high proliferative potential CFCs. The effects of MGDF were more modest on CD38(+)CD34(2+) progenitors with only additive increases in colony formation being observed. These findings suggest that MGDF administration in fetuses and neonates may strongly affect the growth and mobilization of primitive hematopoietic progenitors and that MGDF may find use in the ex vivo growth and expansion of fetal stem cells.

  1. Overexpression of vascular endothelial growth factor accelerates early vascularization and improves healing of genetically modified cultured skin substitutes.

    PubMed

    Supp, Dorothy M; Boyce, Steven T

    2002-01-01

    Cultured skin substitutes (CSS) lack a vascular plexus, leading to slower vascularization after grafting than split-thickness skin autograft. CSS containing keratinocytes genetically modified to overexpress vascular endothelial growth factor (VEGF) were previously shown to exhibit enhanced vascularization up to 2 weeks after grafting to athymic mice. The present study examines whether enhanced vascularization compared with controls persists after stable engraftment is achieved and analyzes VEGF expression, wound contraction, and engraftment. Control and VEGF-modified (VEGF+) CSS were grafted onto full-thickness wounds in athymic mice. VEGF expression was detected in VEGF+ CSS 14 weeks after grafting. Graft contraction was significantly lower in VEGF+ CSS compared with controls, suggesting more stable engraftment and better tissue development. Positive HLA-ABC staining, indicating persistence of human cells, was seen in 86.7% (13/15) of grafted VEGF+ CSS, compared with 58.3% (7/12) of controls. Differences in dermal vascularization between control and VEGF+ grafts were significant 1 week after surgery, but not at later times. However, the distribution of vessels was different, with more vessels in the upper dermis of VEGF+ grafts. These results suggest that VEGF overexpression in genetically modified CSS acts to accelerate early graft vascularization and can contribute to improved healing of full-thickness skin wounds.

  2. Transforming growth factor-beta and its effect on reepithelialization of partial-thickness ear wounds in transgenic mice.

    PubMed

    Tredget, Eric B; Demare, Jack; Chandran, Geethan; Tredget, Edward E; Yang, Liju; Ghahary, Aziz

    2005-01-01

    Transforming growth factor-beta (TGF-beta) is known to affect nearly every aspect of wound repair. Many of the effects have been extensively investigated; however, the primary effect of endogenously derived TGF-beta on wound reepithelialization is still not completely understood. To examine this, two types of wounds were made on a transgenic mouse over-expressing TGF-beta1. Full-thickness back wounds were made to compare the wound healing process in the presence of compensatory healing mechanisms. Superficial partial-thickness ear wounds involving only the epidermis were made to determine the effect of TGF-beta on reepithelialization. In the partial-thickness ear wounds, at later time points, the transgenic group had smaller epithelial gaps than the wild-type mice. A greater number of actively proliferating cells, as determined by bromodeoxyuridine incorporation, was also found in the transgenic mice at post-injury day 8. These results show that TGF-beta1 stimulates the rate of reepithelialization at later time points in partial-thickness wounds. However, in the full-thickness back wounds, the transgenic animals exhibited a slower reepithelialization rate at all time points and the number of bromodeoxyuridine-positive cells was fewer. Our findings would suggest that the overexpression of TGF-beta1 speeds the rate of wound closure in partial-thickness wounds by promoting keratinocyte migration. In full-thickness wounds, however, the overexpression of TGF-beta1 slows the rate of wound reepithelialization.

  3. Stimulation of body weight increase and epiphyseal cartilage growth by insulin like growth factor

    NASA Technical Reports Server (NTRS)

    Ellis, S.

    1981-01-01

    The ability of insulin-like growth factor (IGF) to induce growth in hypophysectomized immature rats was tested by continuous infusion of the partially purified factor at daily doses of 6, 21, and 46 mU for an 8-day period. A dose-dependent growth of the proximal epiphyseal cartilage of the tibia and an associated stimulation of the primary spongiosa were produced by these amounts of IGF. The two highest doses of IGF also resulted in dose-dependent increases of body weight. Gel permeation of the sera at neutrality showed that the large-molecular-weight IGF binding protein was not induced by the infusion of IGF, whereas it ws generated in the sera of hypophysectomized rats that were infused with daily doses of 86 mU of human growth hormone.

  4. Induction of nerve growth factor receptors on cultured human melanocytes

    SciTech Connect

    Peacocke, M.; Yaar, M.; Mansur, C.P.; Chao, M.V.; Gilchrest, B.A. )

    1988-07-01

    Normal differentiation and malignant transformation of human melanocytes involve a complex series of interactions during which both genetic and environmental factors play roles. At present, the regulation of these processes is poorly understood. The authors have induced the expression of nerve growth factor (NGF) receptors on cultured human melanocytes with phorbol 12-tetradecanoate 13-acetate and have correlated this event with the appearance of a more differentiated, dendritic morphology. Criteria for NGF receptor expression included protein accumulation and cell-surface immunofluorescent staining with a monoclonal antibody directed against the human receptor and induction of the messenger RNA species as determined by blot-hybridization studies. The presence of the receptor could also be induced by UV irradiation or growth factor deprivation. The NGF receptor is inducible in cultured human melanocytes, and they suggest that NGF may modulate the behavior of this neural crest-derived cell in the skin.

  5. Hematopoietic growth factors in drug-induced agranulocytosis.

    PubMed

    Pavithran, K; Thomas, M

    2002-05-01

    Drug-induced agranulocytosis (DIA) is a potentially fatal disorder. Hematopoietic growth factors have been used in the treatment of DIA. We report nine cases of DIA treated with granulocyte macrophage - colony stimulating factor (GM-CSF) in a dose of 300 microg/day. All the patients had evidence of systemic infection. Mean time to reach an absolute neutrophil count of 0.5 x 10(9)/L was three days. One patient succumbed to the disease. The cause of death was multiorgan failure. No adverse events were observed with GM-CSF. We conclude that hematopoietic growth factors are useful in shortening the period of neutropenia and reducing morbidity and mortality in these patients.

  6. Extrinsic Factors Influencing Fetal Deformations and Intrauterine Growth Restriction

    PubMed Central

    Moh, Wendy; Graham, John M.; Wadhawan, Isha; Sanchez-Lara, Pedro A.

    2012-01-01

    The causes of intrauterine growth restriction (IUGR) are multifactorial with both intrinsic and extrinsic influences. While many studies focus on the intrinsic pathological causes, the possible long-term consequences resulting from extrinsic intrauterine physiological constraints merit additional consideration and further investigation. Infants with IUGR can exhibit early symmetric or late asymmetric growth abnormality patterns depending on the fetal stage of development, of which the latter is most common occurring in 70–80% of growth-restricted infants. Deformation is the consequence of extrinsic biomechanical factors interfering with normal growth, functioning, or positioning of the fetus in utero, typically arising during late gestation. Biomechanical forces play a critical role in the normal morphogenesis of most tissues. The magnitude and direction of force impact the form of the developing fetus, with a specific tissue response depending on its pliability and stage of development. Major uterine constraining factors include primigravida, small maternal size, uterine malformation, uterine fibromata, early pelvic engagement of the fetal head, aberrant fetal position, oligohydramnios, and multifetal gestation. Corrective mechanical forces similar to those that gave rise to the deformation to reshape the deformed structures are often used and should take advantage of the rapid postnatal growth to correct form. PMID:22888434

  7. Human epidermal growth factor and the proliferation of human fibroblasts.

    PubMed

    Carpenter, G; Cohen, S

    1976-06-01

    The effect of human epidermal growth factor (hEGF), a 5,400 molecular weight polypeptide isolated from human urine, on the growth of human foreskin fibroblasts (HF cells) was studied by measuring cell numbers and the incorporation of labeled thymidine. The addition of hEGF to HF cells growing in a medium containing 10% calf serum resulted in a 4-fold increase in the final density. The presence of hEGF also promoted the growth of HF cells in media containing either 1% calf serum or 10% gamma globulin-free serum. The addition of hEGF to quiescent confluent monolayers of HF cells, maintained in a medium with 1% calf serum for 48 hours, resulted in a 10- to 20-fold increase in the amount of 3H-thymidine incorporation after 20-24 hours. The stimulation of thymidine incorporation was maximal at an hEGF concentration of 2 ng/ml, was dependent on the presence of serum, and was enhanced by the addition of ascorbic acid. In confluent cultures of HF cells, subject to density dependent inhibition of growth, hEGF was able to stimulate DNA synthesis more effectively than fresh calf serum. Human EGF stimulated DNA synthesis in quiescent cultures, however, regardless of cell density. The addition of rabbit anti-hEGF inhibited all effects of this growth factor on HF cells.

  8. Vascular Endothelial Growth Factor Acts Primarily via Platelet-Derived Growth Factor Receptor α to Promote Proliferative Vitreoretinopathy

    PubMed Central

    Pennock, Steven; Haddock, Luis J.; Mukai, Shizuo; Kazlauskas, Andrius

    2015-01-01

    Proliferative vitreoretinopathy (PVR) is a nonneovascular blinding disease and the leading cause for failure in surgical repair of rhegmatogenous retinal detachments. Once formed, PVR is difficult to treat. Hence, there is an acute interest in developing approaches to prevent PVR. Of the many growth factors and cytokines that accumulate in vitreous as PVR develops, neutralizing vascular endothelial growth factor (VEGF) A has recently been found to prevent PVR in at least one animal model. The goal of this study was to test if Food and Drug Administration–approved agents could protect the eye from PVR in multiple animal models and to further investigate the underlying mechanisms. Neutralizing VEGF with aflibercept (VEGF Trap-Eye) safely and effectively protected rabbits from PVR in multiple models of disease. Furthermore, aflibercept reduced the bioactivity of both experimental and clinical PVR vitreous. Finally, although VEGF could promote some PVR-associated cellular responses via VEGF receptors expressed on the retinal pigment epithelial cells that drive this disease, VEGF's major contribution to vitreal bioactivity occurred via platelet-derived growth factor receptor α. Thus, VEGF promotes PVR by a noncanonical ability to engage platelet-derived growth factor receptor α. These findings indicate that VEGF contributes to nonangiogenic diseases and that anti–VEGF-based therapies may be effective on a wider spectrum of diseases than previously appreciated. PMID:25261788

  9. Nutrition and the insulin-like growth factor system.

    PubMed

    Estívariz, C F; Ziegler, T R

    1997-08-01

    Nutritional status is a key regulator of the circulating and tissue insulin-like growth factor (IGF) system. IGF-I mRNA and protein levels decrease in tissues such as liver and intestine with fasting and are restored with refeeding. Additional studies suggest that the level of protein and calorie intake independently regulate plasma IGF-I concentrations in man. The level of nutrition effects the biological actions of recombinant growth hormone (GH) and IGF-I administration in humans. Limited data demonstrate that plasma and tissue levels of the insulin-like growth factor binding proteins (IGFBPs) are also sensitive to nutrient intake. Specific micronutrients, such as potassium, magnesium and zinc also appear to be important for optimal IGF-I synthesis and anabolic effects in animal models. Malnutrition is common in elderly patients, however, the interaction between specific nutrients, general nutritional status and the aging process on the IGF system is incompletely understood. Mechanisms of nutrient-IGF system interactions which may affect the biological actions of IGF-I, IGF-II, and the IGFBPs are increasingly being determined in basic studies. The effects of underlying nutritional status and responses to dietary intake will be important to evaluate in clinical studies of the IGF system and exogenous growth factor therapy.

  10. Transforming growth factor (TGF)-. alpha. in human milk

    SciTech Connect

    Okada, Masaki; Wakai, Kae; Shizume, Kazuo ); Iwashita, Mitsutoshi ); Ohmura, Eiji; Kamiya, Yoshinobu; Murakami, Hitomi; Onoda, Noritaka; Tsushima, Toshio

    1991-01-01

    Transforming growth factor (TGF)-{alpha} and epidermal growth factor (EGF) were measured in human milk by means of homologous radioimmunoassay. As previously reported, EGF concentration in the colostrum was approximately 200 ng/ml and decreased to 50 ng/ml by day 7 postpartum. The value of immunoreactive (IR)-TGF-{alpha} was 2.2-7.2 ng/ml, much lower than that of EGF. In contrast to EGF, the concentration of IR-TGF-{alpha} was fairly stable during the 7 postpartum days. There was no relationship between the concentrations of IR-TGF-{alpha} and IR-EGF, suggesting that the regulatory mechanism in the release of the two growth factors is different. On gel-chromatography using a Sephadex G-50 column, IR-EGF appeared in the fraction corresponding to that of authentic human EGF, while 70%-80% of the IR-TGF-{alpha} was eluted as a species with a molecular weight greater than that of authentic human TGF-{alpha}. Although the physiological role of TGF-{alpha} in milk is not known, it is possible that it is involved in the development of the mammary gland and/or the growth of newborn infants.

  11. Vascular growth factors and receptors in capillary hemangioblastomas and hemangiopericytomas.

    PubMed Central

    Hatva, E.; Böhling, T.; Jääskeläinen, J.; Persico, M. G.; Haltia, M.; Alitalo, K.

    1996-01-01

    Capillary hemangioblastomas and hemangiopericytomas are highly vascular central nervous system tumors of controversial origin. Of interest in their pathogenesis are mechanisms regulating endothelial cell growth. The endothelial cell mitogen vascular endothelial growth factor (VEGF) stimulates angiogenesis, and together with its two receptor tyrosine kinases VEGFR-1(FLT1) and VEGFR-2(KDR), is up-regulated during the malignant progression of gliomas. We have analyzed the expression of VEGF and its receptors, the related placental growth factor (PlGF) and the endothelial receptors FLT4 and Tie by in situ hybridization in capillary hemangioblastomas and hemangiopericytomas. VEGF mRNA was up-regulated in all of the hemangiopericytomas studied and highly expressed in the stromal cells of hemangioblastomas. In addition, some hemangioblastoma tumor cells expressed high levels of PlGF. Significantly elevated levels of Tie mRNA, Tie protein, VEGFR-1, and VEGFR-2 but not FLT4 mRNAs were observed in the endothelia of both tumor types. In hemangioblastomas, however, the receptors were also highly expressed by a subpopulation of stromal cells. Consistent results were obtained for a human hemangioblastoma cell line in culture. Up-regulation of the endothelial growth factors and receptors may result in autocrine or paracrine stimulation of endothelial cells and their precursors involved in the genesis of these two vascular tumors. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8774132

  12. Therapeutic Targeting of Fibroblast Growth Factor Receptors in Gastric Cancer

    PubMed Central

    Fujimori, Yoshitaka; Otsuki, Sho; Sato, Yuya; Nakagawa, Masatoshi

    2015-01-01

    Chemotherapy has become the global standard treatment for patients with metastatic or unresectable gastric cancer (GC), although outcomes remain unfavorable. Many molecular-targeted therapies inhibiting signaling pathways of various tyrosine kinase receptors have been developed, and monoclonal antibodies targeting human epidermal growth factor receptor 2 (HER2) have become standard therapy for HER2-positive GC. An inhibitor of vascular endothelial growth factor receptor 2 or MET has also produced promising results in patients with GC. Fibroblast growth factor receptors (FGFR) play key roles in tumor growth via activated signaling pathways in GC. Genomic amplification of FGFR2 leads to the aberrant activation found in GC tumors and is related to survival in patients with GC. This review discusses the clinical relevance of FGFR in GC and examines FGFR as a potential therapeutic target in patients with GC. Preclinical studies in animal models suggest that multitargeted tyrosine kinase inhibitors (TKIs), including FGFR inhibitor, suppress tumor cell proliferation and delay tumor progression. Several TKIs are now being evaluated in clinical trials as treatment for metastatic or unresectable GC harboring FGFR2 amplification. PMID:26000013

  13. FGF19 functions as autocrine growth factor for hepatoblastoma

    PubMed Central

    Elzi, David J.; Song, Meihua; Blackman, Barron; Weintraub, Susan T.; López-Terrada, Dolores; Chen, Yidong; Tomlinson, Gail E.; Shiio, Yuzuru

    2016-01-01

    Hepatoblastoma is the most common liver cancer in children, accounting for over 65% of all childhood liver malignancies. Hepatoblastoma is distinct from adult liver cancer in that it is not associated with hepatitis virus infection, cirrhosis, or other underlying liver pathology. The paucity of appropriate cell and animal models has been hampering the mechanistic understanding of hepatoblastoma pathogenesis. Consequently, there is no molecularly targeted therapy for hepatoblastoma. To gain insight into cytokine signaling in hepatoblastoma, we employed mass spectrometry to analyze the proteins secreted from Hep293TT hepatoblastoma cell line we established and identified the specific secretion of fibroblast growth factor 19 (FGF19), a growth factor for liver cells. We determined that silencing FGF19 by shRNAs or neutralizing secreted FGF19 by anti-FGF19 antibody inhibits the proliferation of hepatoblastoma cells. Furthermore, blocking FGF19 signaling by an FGF receptor kinase inhibitor suppressed hepatoblastoma growth. RNA expression analysis in hepatoblastoma tumors revealed that the high expression of FGF19 signaling pathway components as well as the low expression of FGF19 signaling repression targets correlates with the aggressiveness of the tumors. These results suggest the role of FGF19 as autocrine growth factor for hepatoblastoma. PMID:27382436

  14. Lifetime growth in wild meerkats: incorporating life history and environmental factors into a standard growth model.

    PubMed

    English, Sinéad; Bateman, Andrew W; Clutton-Brock, Tim H

    2012-05-01

    Lifetime records of changes in individual size or mass in wild animals are scarce and, as such, few studies have attempted to model variation in these traits across the lifespan or to assess the factors that affect them. However, quantifying lifetime growth is essential for understanding trade-offs between growth and other life history parameters, such as reproductive performance or survival. Here, we used model selection based on information theory to measure changes in body mass over the lifespan of wild meerkats, and compared the relative fits of several standard growth models (monomolecular, von Bertalanffy, Gompertz, logistic and Richards). We found that meerkats exhibit monomolecular growth, with the best model incorporating separate growth rates before and after nutritional independence, as well as effects of season and total rainfall in the previous nine months. Our study demonstrates how simple growth curves may be improved by considering life history and environmental factors, which may be particularly relevant when quantifying growth patterns in wild populations. PMID:22108854

  15. Very-high-growth-factor Planar Ablative Rayleigh Taylor Experiments

    SciTech Connect

    Bradley, D K; Braun, D G; Glendinning, S G; Edwards, M J; Milovich, J L; Sorce, C M; Collins, G W; Haan, S W; Page, R H

    2006-10-30

    The Rayleigh-Taylor (RT) instability is an important factor in bounding the performance envelope of ignition targets. This paper describes an experiment for ablative RT instability that for the first time achieves growth factors close to those expected to occur in ignition targets at the National Ignition Facility (NIF). The large growth allows small seed perturbations to be detected and can be used to place an upper bound on perturbation growth at the ablation front resulting from microstructure in the preferred Be ablator. The experiments were performed on the Omega laser using a halfraum 1.2 mm long by 2 mm diameter with a 75% laser entrance hole. The halfraum was filled with {approx} 1 atm of neopentane to delay gold plasma from closing the diagnostic line of sight down the axis of the halfraum. The ablator was mounted at the base of the halfraum, and was accelerated by a two stepped X-ray pulse consisting of an early time section {approx} 100 eV to emulate the NIF foot followed by an approximately constant {approx} 150 eV drive sustained over an additional 5-7ns. It is this long pulse duration and late time observation that distinguishes the present work from previous experiments, and is responsible for the large growth that is achieved. The growth of a 2D sinusoidal perturbation machined on the drive side of the ablator was measured using face-on radiography. The diagnostic view remained open until {approx} 11 ns with maximum growth factors measured to be {approx} 200. The trajectory of the ablator was measured using streaked backlit radiography. The design and analysis of the experiments is described, and implications for experiments on ignition target ablators are discussed.

  16. HPV-18 immortalization of human keratinocytes.

    PubMed

    Kaur, P; McDougall, J K

    1989-11-01

    The oncogenic potential of human papillomavirus type 18 which is found in a significant number of cervical and penile cancer biopsies was tested in primary human keratinocytes derived from neonatal foreskin. Viral DNA and a gene for resistance to neomycin were introduced into these cells by calcium phosphate transfection. Selection of cells in G418 led to the isolation of resistant colonies which were propagated in culture. Four cell lines termed FE-A, FEH 18L, FEP18-5, and FEP18-11 have been maintained in culture for 1 1/2-2 years and were selected for further analysis. In all cases the viral DNA was integrated into the cellular genome and the early genes were transcribed, including RNA complementary to the E2, E6, and E7 open reading frames. Radioimmunoprecipitation showed that all cell lines synthesized the E6 and E7 proteins. However, none of the cell lines tested were tumorigenic. The differentiation capacity of these cells was analyzed by assessing their ability to proliferate clonally after exposure to 1.2 mM calcium chloride. All four cell lines were resistant to this stimulus and formed colonies upon return to regular growth medium whereas normal cells differentiated terminally. K6a and K14 keratin RNA expression was down-regulated in the HPV immortalized cell lines compared to primary human epithelial cells.

  17. GH responses to growth hormone releasing factor in depression.

    PubMed

    Thomas, R; Beer, R; Harris, B; John, R; Scanlon, M

    1989-01-01

    The growth hormone (GH), thyrotrophin (TSH) and prolactin response to growth hormone releasing factor (GRF) was investigated in 18 patients suffering from major depression with melancholia and in 18 age- and sex-matched normal controls. There was no significant difference in the GH response to GRF stimulation between the patients and controls and in neither subject group was there a demonstrable TSH or prolactin response to GRF. These findings indicate that the pathophysiology underlying the blunted GH response to pharmacological challenge, demonstrated in other studies, must lie at a suprapituitary level.

  18. Expression of neu protein, epidermal growth factor receptor, and transforming growth factor alpha in breast cancer. Correlation with clinicopathologic parameters.

    PubMed Central

    Lundy, J.; Schuss, A.; Stanick, D.; McCormack, E. S.; Kramer, S.; Sorvillo, J. M.

    1991-01-01

    The major objectives of this study were twofold: to determine 1) if growth factors or growth factor receptors were expressed similarly or differently in a clinically well-characterized group of breast cancer patients and 2) if these phenotypic characteristics were associated with any of the commonly used prognostic parameters. Formalin-fixed paraffin-embedded tumor tissue from 51 node-positive breast cancer patients were analyzed for the expression of neu, epidermal growth factor-receptor (EGF-R), and transforming growth factor alpha (TGF alpha) using immunoperoxidase staining. Positive membranous staining for neu was observed in 15 (29%) tumors. Over-expression of neu was observed in high-grade, estrogen-receptor-negative tumors (P less than 0.05). Epidermal growth factor receptor was expressed in 22 (43%) of the tumors analyzed and found to a greater degree in estrogen-receptor-negative and high-grade tumors (P less than 0.025). A significant correlation between neu and EGF-R expression was also noted. Tumors expressing membranous staining of neu had a greater than 70% chance of expressing EGF-R (P less than 0.01). Expression of TGF alpha was found in 68% of tumors and TGF alpha was detected in grade 1 and 2 tumor to a greater degree than EGF-R. The authors conclude that assaying tumors for these antigens may give additional phenotypic characteristics that can give further insight into the biology of breast cancer. Images Figure 1 Figure 2 Figure 3 PMID:1711294

  19. Divergent effects of epidermal growth factor and transforming growth factors on a human endometrial carcinoma cell line.

    PubMed

    Korc, M; Haussler, C A; Trookman, N S

    1987-09-15

    Epidermal growth factor (EGF), at concentrations ranging from 0.83 to 4.98 nM, markedly inhibited the proliferation of RL95-2 cells that were seeded at low plating densities (4.7 X 10(3) cells/cm2). Under the same incubation conditions, 16.6 pM EGF enhanced cell proliferation. At high plating densities (2.5 X 10(4) cells/cm2) 0.83 nM EGF also stimulated cell proliferation. Both the inhibitory and stimulatory effects of EGF were mimicked by transforming growth factor-alpha (TGF-alpha). However, the inhibitory action of TGF-alpha was always greater that of EGF. Binding studies with 125I-labeled TGF-alpha indicated that maximal cell surface binding of TGF-alpha occurred at 15 min, whereas maximal internalization occurred at 45 min. Both cell surface and internalized radioactivity declined sharply thereafter. Analysis of radioactivity released into the incubation medium during pulse-chase experiments indicated that RL95-2 cells extensively degraded both TGF-alpha and EGF. The lysosomotropic compound methylamine arrested the generation of low-molecular-weight degradation products of EGF, but not of TGF-alpha. In contrast to EGF and TGF-alpha, transforming growth factor-beta (TGF-beta) inhibited the proliferation of RL95-2 cells that were seeded at either low or high plating densities. Further, transforming growth factor-beta induced the appearance of large cuboidal cells that were readily distinguished from cells treated with either EGF or TGF-alpha. These findings point to complex regulatory actions of growth factors on the proliferation of RL95-2 cells and suggest that the processing of TGF-alpha following EGF receptor activation is distinct from the processing of EGF. PMID:3497713

  20. Insulin-like growth factor II mediates epidermal growth factor-induced mitogenesis in cervical cancer cells.

    PubMed Central

    Steller, M A; Delgado, C H; Zou, Z

    1995-01-01

    There is increasing evidence that activation of the insulin-like growth factor I (IGF-I) receptor plays a major role in the control of cellular proliferation of many cell types. We studied the mitogenic effects of IGF-I, IGF-II, and epidermal growth factor (EGF) on growth-arrested HT-3 cells, a human cervical cancer cell line. All three growth factors promoted dose-dependent increases in cell proliferation. In untransformed cells, EGF usually requires stimulation by a "progression" factor such as IGF-I, IGF-II, or insulin (in supraphysiologic concentrations) in order to exert a mitogenic effect. Accordingly, we investigated whether an autocrine pathway involving IGF-I or IGF-II participated in the EGF-induced mitogenesis of HT-3 cells. With the RNase protection assay, IGF-I mRNA was not detected. However, IGF-II mRNA increased in a time-dependent manner following EGF stimulation. The EGF-induced mitogenesis was abrogated in a dose-dependent manner by IGF-binding protein 5 (IGFBP-5), which binds to IGF-II and neutralizes it. An antisense oligonucleotide to IGF-II also inhibited the proliferative response to EGF. In addition, prolonged, but not short-term, stimulation with EGF resulted in autophosphorylation of the IGF-I receptor, and coincubations with both EGF and IGFBP-5 attenuated this effect. These data demonstrate that autocrine secretion of IGF-II in HT-3 cervical cancer cells can participate in EGF-induced mitogenesis and suggest that autocrine signals involving the IGF-I receptor occur "downstream" of competence growth factor receptors such as the EGF receptor. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8618825

  1. Growth factor choice is critical for successful functionalization of nanoparticles

    PubMed Central

    Pinkernelle, Josephine; Raffa, Vittoria; Calatayud, Maria P.; Goya, Gerado F.; Riggio, Cristina; Keilhoff, Gerburg

    2015-01-01

    Nanoparticles (NPs) show new characteristics compared to the corresponding bulk material. These nanoscale properties make them interesting for various applications in biomedicine and life sciences. One field of application is the use of magnetic NPs to support regeneration in the nervous system. Drug delivery requires a functionalization of NPs with bio-functional molecules. In our study, we functionalized self-made PEI-coated iron oxide NPs with nerve growth factor (NGF) and glial cell-line derived neurotrophic factor (GDNF). Next, we tested the bio-functionality of NGF in a rat pheochromocytoma cell line (PC12) and the bio-functionality of GDNF in an organotypic spinal cord culture. Covalent binding of NGF to PEI-NPs impaired bio-functionality of NGF, but non-covalent approach differentiated PC12 cells reliably. Non-covalent binding of GDNF showed a satisfying bio-functionality of GDNF:PEI-NPs, but turned out to be unstable in conjugation to the PEI-NPs. Taken together, our study showed the importance of assessing bio-functionality and binding stability of functionalized growth factors using proper biological models. It also shows that successful functionalization of magnetic NPs with growth factors is dependent on the used binding chemistry and that it is hardly predictable. For use as therapeutics, functionalization strategies have to be reproducible and future studies are needed. PMID:26388717

  2. Juvenile nasopharyngeal angiofibroma contain an angiogenic growth factor: basic FGF.

    PubMed

    Schiff, M; Gonzalez, A M; Ong, M; Baird, A

    1992-08-01

    The presence of an angiogenic protein basic fibroblast growth factor (FGF) was established in juvenile nasopharyngeal angiofibroma (JNF). Extracts of these tumors have the capacity to stimulate endothelial cell proliferation. This activity is indistinguishable from basic FGF. The biological activity contained in the extracts binds to heparin-Sepharose columns and is eluted with a characteristic 2 mol sodium chloride. The exact fraction of the biological activity corresponds to the location where an immunoreactive basic FGF can be detected by radioimmunoassay. These same fractions contain an 18,000-d molecule which is identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with an antibody to basic FGF. Indeed, immunohistochemical studies localize the growth factor to the endothelium of JNF. Although these findings do not establish that basic FGF mediates the development of this angiofibroma, they do support the possibility that the pathogenesis of JNF is associated with the presence of angiogenic factors like basic FGF. If this is the case, a comprehensive study of the etiology of JNF may lead to a better understanding of how locally produced growth factors mediate proliferative disease and how its modification might lead to better treatment on a biological basis.

  3. Key roles of necroptotic factors in promoting tumor growth

    PubMed Central

    Liu, Xinjian; Zhou, Min; Mei, Ling; Ruan, Jiaying; Hu, Qian; Peng, Jing; Su, Hang; Liao, Hong; Liu, Shanling; Liu, WeiPing; Wang, He; Huang, Qian; Li, Fang; Li, Chuan-Yuan

    2016-01-01

    Necroptotic factors are generally assumed to play a positive role in tumor therapy by eliminating damaged tumor cells. Here we show that, contrary to expectation, necroptotic factors RIPK1, RIPK3, and MLKL promote tumor growth. We demonstrate that genetic knockout of necroptotic genes RIPK1, RIPK3, or MLKL in cancer cells significantly attenuated their abilities to grow in an anchorage-independent manner. In addition, they exhibited significantly enhanced radiosensitivity. The knockout cells also showed greatly reduced ability to form tumors in mice. Moreover, necrosulfonamide (NSA), a previously identified chemical inhibitor of necroptosis, could significantly delay tumor growth in a xenograft model. Mechanistically, we show that necroptoic factors play a significant role in maintaining the activity of NF-κB. Finally, we found that high levels of phosphorylated MLKL in human esophageal and colon cancers are associated with poor overall survival. Taken together, we conclude that pro-necroptic factors such as RIPK1, RIPK3, and MLKL may play a role in supporting tumor growth, and MLKL may be a promising target for cancer treatment. PMID:26959742

  4. MiR-146a negatively regulates TLR2-induced inflammatory responses in keratinocytes.

    PubMed

    Meisgen, Florian; Xu Landén, Ning; Wang, Aoxue; Réthi, Bence; Bouez, Charbel; Zuccolo, Michela; Gueniche, Audrey; Ståhle, Mona; Sonkoly, Enikö; Breton, Lionel; Pivarcsi, Andor

    2014-07-01

    Keratinocytes represent the first line of defense against pathogens in the skin and have important roles in initiating and regulating inflammation during infection and autoimmunity. Here we investigated the role of miR-146a in the regulation of the innate immune response of keratinocytes. Toll-like receptor 2 (TLR2) stimulation of primary human keratinocytes resulted in an NF-κB- and mitogen-activated protein kinase-dependent upregulation of miR-146a expression, which was surprisingly long lasting, contrasting with the rapid and transient induction of inflammatory mediators. Overexpression of miR-146a significantly suppressed the production of IL-8, CCL20, and tumor necrosis factor-α, which functionally suppressed the chemotactic attraction of neutrophils by keratinocytes. Inhibition of endogenous miR-146a induced the production of inflammatory mediators even in nonstimulated keratinocytes, and potentiated the effect of TLR2 stimulation. Transcriptomic profiling revealed that miR-146a suppresses the expression of a large number of immune-related genes in keratinocytes. MiR-146a downregulated interleukin-1 receptor-associated kinase 1 and TNF receptor-associated factor 6, two key adapter molecules downstream of TLR signaling, and suppressed NF-κB promoter-binding activity as shown by promoter luciferase experiments. Together, these data identify miR-146a as a regulatory element in keratinocyte innate immunity, which prevents the production of inflammatory mediators under homeostatic conditions and serves as a potent negative feedback regulator after TLR2 stimulation. PMID:24670381

  5. Nrf2 Regulates the Sensitivity of Mouse Keratinocytes to Nitrogen Mustard via Multidrug Resistance-Associated Protein 1 (Mrp1).

    PubMed

    Udasin, Ronald G; Wen, Xia; Bircsak, Kristin M; Aleksunes, Lauren M; Shakarjian, Michael P; Kong, Ah-Ng Tony; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

    2016-01-01

    Sulfur mustard and nitrogen mustard (mechlorethamine, HN2) are potent vesicants developed as chemical warfare agents. These electrophilic, bifunctional alkylating agents cause skin injury, including inflammation, edema, and blistering. HN2 covalently modifies macromolecules such as DNA, RNA, and proteins or is scavenged by glutathione, forming adducts that can contribute to toxicity. Multidrug resistance-associated protein 1 (Mrp1/MRP1) is a transmembrane ATPase known to efflux glutathione-conjugated electrophiles. In the present studies, we examined the effects of modulating Mrp1-mediated transport activity on the sensitivity of primary and PAM212 mouse keratinocytes to HN2. Primary keratinocytes, and to a lesser extent, PAM212 cells, express Mrp1 mRNA and protein and possess Mrp1 functional activity, as measured by calcein efflux. Sulforaphane, an activator of Nrf2, increased Mrp1 mRNA, protein, and functional activity in primary keratinocytes and PAM212 cells and decreased their sensitivity to HN2-induced growth inhibition (IC(50) = 1.4 and 4.8 µM in primary keratinocytes and 1 and 13 µM in PAM212 cells, in the absence and presence of sulforaphane, respectively). The Mrp1 inhibitor, MK-571, reversed the effects of sulforaphane on HN2-induced growth inhibition in both primary keratinocytes and PAM212 cells. In primary keratinocytes from Nrf2(-/-) mice, sulforaphane had no impact on Mrp1 expression or activity, or on sensitivity to HN2, demonstrating that its effects depend on Nrf2. These data suggest that Mrp1-mediated efflux is important in regulating HN2-induced keratinocyte growth inhibition. Enhancing HN2 efflux from keratinocytes may represent a novel strategy for mitigating vesicant-induced cytotoxicity. PMID:26454883

  6. Downregulation of TNIP1 Expression Leads to Increased Proliferation of Human Keratinocytes and Severer Psoriasis-Like Conditions in an Imiquimod-Induced Mouse Model of Dermatitis.

    PubMed

    Chen, Yan; Yan, Heng; Song, Zhiqiang; Chen, Fangru; Wang, Huan; Niu, Jun; Shi, Xiaowei; Zhang, Dongmei; Zhang, Na; Zhai, Zhifang; Zhong, Baiyu; Cheng, Liangjin; Qian, Tian; Hao, Fei

    2015-01-01

    Psoriasis is a chronic, inflammatory skin disease involving both environmental and genetic factors. According to genome-wide association studies (GWAS), the TNIP1 gene, which encodes the TNF-α-induced protein 3-interacting protein 1 (TNIP1), is strongly linked to the susceptibility of psoriasis. TNIP1 is a widely expressed ubiquitin sensor that binds to the ubiquitin-editing protein A20 and restricts TNF- and TLR-induced signals. In our study, TNIP1 expression decreased in specimens of epidermis affected by psoriasis. Based on previous studies suggesting a role for TNIP1 in modulating cancer cell growth, we investigated its role in keratinocyte proliferation, which is clearly abnormal in psoriasis. To mimic the downregulation or upregulation of TNIP1 in HaCaT cells and primary human keratinocytes (PHKs), we used a TNIP1 specific small interfering hairpin RNA (TNIP1 shRNA) lentiviral vector or a recombinant TNIP1 (rTNIP1) lentiviral vector, respectively. Blocking TNIP1 expression increased keratinocyte proliferation, while overexpression of TNIP1 decreased keratinocyte proliferation. Furthermore, we showed that TNIP1 signaling might involve extracellular signal-regulated kinase1/2 (Erk1/2) and CCAAT/enhancer-binding protein β (C/EBPβ) activity. Intradermal injection of TNIP1 shRNA in BALB/c mice led to exaggerated psoriatic conditions in imiquimod (IMQ)-induced psoriasis-like dermatitis. These findings indicate that TNIP1 has a protective role in psoriasis and therefore could be a promising therapeutic target. PMID:26046540

  7. Impact of epidermal growth factor receptor and transforming growth factor-α on hepatitis C virus-induced hepatocarcinogenesis.

    PubMed

    Badawy, Afkar Abdel-Ghany; El-Hindawi, Ali; Hammam, Olfat; Moussa, Mona; Gabal, Samia; Said, Noha

    2015-10-01

    Epidermal growth factor receptor system plays a central hepato-protective and pro-regenerative role in liver. Transforming growth factor-α (TGF-α) is an important autocrine growth regulator of hepatocytes that plays a role in development of hepatocellular carcinoma (HCC) among patients with chronic hepatitis C (CHC). This study was done on 40 core liver biopsies from patients with CHC, 20 liver specimens from HCC cases on top of CHC as well as five normal controls. All were immunohistochemically stained with epidermal growth factor receptor (EGFR) and TGF-α antibodies. Some selected HCC cases were submitted for FISH technique to detect EGFR gene alteration. By immunohistochemistry EGFR and TGF-α were overexpressed in HCC and cirrhotic cases compared to CHC cases without cirrhosis. Also, their expression was stronger in CHC cases with higher grades of activity and stages of fibrosis compared to lower ones. FISH positive results for EGFR were detected in 33.3% of the examined HCC cases. EGFR and TGF-α can be used as predictive markers for activity, fibrosis, and carcinogenesis in CHC patients. Overexpression of EGFR in HCC patients can be promising in selecting those who can get benefit from anti-EGFR target therapy. PMID:26279457

  8. Insulin-like growth factors in embryonic and fetal growth and skeletal development (Review).

    PubMed

    Agrogiannis, Georgios D; Sifakis, Stavros; Patsouris, Efstratios S; Konstantinidou, Anastasia E

    2014-08-01

    The insulin-like growth factors (IGF)-I and -II have a predominant role in fetal growth and development. IGFs are involved in the proliferation, differentiation and apoptosis of fetal cells in vitro and the IGF serum concentration has been shown to be closely correlated with fetal growth and length. IGF transcripts and peptides have been detected in almost every fetal tissue from as early in development as pre‑implantation to the final maturation stage. Furthermore, IGFs have been demonstrated to be involved in limb morphogenesis. However, although ablation of Igf genes in mice resulted in growth retardation and delay in skeletal maturation, no impact on outgrowth and patterning of embryonic limbs was observed. Additionally, various molecular defects in the Igf1 and Igf1r genes in humans have been associated with severe intrauterine growth retardation and impaired skeletal maturation, but not with truncated limbs or severe skeletal dysplasia. The conflicting data between in vitro and in vivo observations with regard to bone morphogenesis suggests that IGFs may not be the sole trophic factors involved in fetal skeletal growth and that redundant mechanisms may exist in chondro- and osteogenesis. Further investigation is required in order to elucidate the functions of IGFs in skeletal development.

  9. Myoferlin is required for insulin-like growth factor response and muscle growth.

    PubMed

    Demonbreun, Alexis R; Posey, Avery D; Heretis, Konstantina; Swaggart, Kayleigh A; Earley, Judy U; Pytel, Peter; McNally, Elizabeth M

    2010-04-01

    Insulin-like growth factor (IGF) is a potent stimulus of muscle growth. Myoferlin is a membrane-associated protein important for muscle development and regeneration. Myoferlin-null mice have smaller muscles and defective myoblast fusion. To understand the mechanism by which myoferlin loss retards muscle growth, we found that myoferlin-null muscle does not respond to IGF1. In vivo after IGF1 infusion, control muscle increased myofiber diameter by 25%, but myoferlin-null muscle was unresponsive. Myoblasts cultured from myoferlin-null muscle and treated with IGF1 also failed to show the expected increase in fusion to multinucleate myotubes. The IGF1 receptor colocalized with myoferlin at sites of myoblast fusion. The lack of IGF1 responsiveness in myoferlin-null myoblasts was linked directly to IGF1 receptor mistrafficking as well as decreased IGF1 signaling. In myoferlin-null myoblasts, the IGF1 receptor accumulated into large vesicular structures. These vesicles colocalized with a marker of late endosomes/lysosomes, LAMP2, specifying redirection from a recycling to a degradative pathway. Furthermore, ultrastructural analysis showed a marked increase in vacuoles in myoferlin-null muscle. These data demonstrate that IGF1 receptor recycling is required for normal myogenesis and that myoferlin is a critical mediator of postnatal muscle growth mediated by IGF1.-Demonbreun, A. R., Posey, A. D., Heretis, K., Swaggart, K. A., Earley, J. U., Pytel, P., McNally, E. M. Myoferlin is required for insulin-like growth factor response and muscle growth.

  10. Growth Factor Liberation and DPSC Response Following Dentine Conditioning.

    PubMed

    Sadaghiani, L; Gleeson, H B; Youde, S; Waddington, R J; Lynch, C D; Sloan, A J

    2016-10-01

    Liberation of the sequestrated bioactive molecules from dentine by the action of applied dental materials has been proposed as an important mechanism in inducing a dentinogenic response in teeth with viable pulps. Although adhesive restorations and dentine-bonding procedures are routinely practiced, clinical protocols to improve pulp protection and dentine regeneration are not currently driven by biological knowledge. This study investigated the effect of dentine (powder and slice) conditioning by etchants/conditioners relevant to adhesive restorative systems on growth factor solubilization and odontoblast-like cell differentiation of human dental pulp progenitor cells (DPSCs). The agents included ethylenediaminetetraacetic acid (EDTA; 10%, pH 7.2), phosphoric acid (37%, pH <1), citric acid (10%, pH 1.5), and polyacrylic acid (25%, pH 3.9). Growth factors were detected in dentine matrix extracts drawn by EDTA, phosphoric acid, and citric acid from powdered dentine. The dentine matrix extracts were shown to be bioactive, capable of stimulating odontogenic/osteogenic differentiation as observed by gene expression and phenotypic changes in DPSCs cultured in monolayer on plastic. Polyacrylic acid failed to solubilize proteins from powdered dentine and was therefore considered ineffective in triggering a growth factor-mediated response in cells. The study went on to investigate the effect of conditioning dentine slices on growth factor liberation and DPSC behavior. Conditioning by EDTA, phosphoric acid, and citric acid exposed growth factors on dentine and triggered an upregulation in genes associated with mineralized differentiation, osteopontin, and alkaline phosphatase in DPSCs cultured on dentine. The cells demonstrated odontoblast-like appearances with elongated bodies and long extracellular processes extending on dentine surface. However, phosphoric acid-treated dentine appeared strikingly less populated with cells, suggesting a detrimental impact on cell

  11. Fibroblast Growth Factor 23 in Long-Duration Spaceflight

    NASA Technical Reports Server (NTRS)

    Bokhari, R.; Zwart, S. R.; Fields, E.; Heer, M.; Sibonga, J.; Smith, S. M.

    2015-01-01

    Many nutritional factors influence bone, from the basics of calcium and vitamin D, to factors which influence bone through acid/base balance, including protein, sodium, and more. Fibroblast growth factor 23 (FGF23) is a recently identified factor, secreted from osteocytes, which is involved in classic (albeit complex) feedback loops controlling phosphorus homeostasis through both vitamin D and parathyroid hormone (PTH) (1, 2). As osteocytes are gravity sensing cells, it is important to determine if there are changes in FGF23 during spaceflight. In extreme cases, such as chronic kidney disease, FGF23 levels are highly elevated. FGF23 imbalances, secondary to dietary influences, may contribute to skeletal demineralization and kidney stone risk during spaceflight.

  12. Nerve Growth Factor: A Focus on Neuroscience and Therapy

    PubMed Central

    Aloe, Luigi; Rocco, Maria Luisa; Omar Balzamino, Bijorn; Micera, Alessandra

    2015-01-01

    Nerve growth factor (NGF) is the firstly discovered and best characterized neurotrophic factor, known to play a critical protective role in the development and survival of sympathetic, sensory and forebrain cholinergic neurons. NGF promotes neuritis outgrowth both in vivo and in vitro and nerve cell recovery after ischemic, surgical or chemical injuries. Recently, the therapeutic property of NGF has been demonstrated on human cutaneous and corneal ulcers, pressure ulcer, glaucoma, maculopathy and retinitis pigmentosa. NGF eye drops administration is well tolerated, with no detectable clinical evidence of systemic or local adverse effects. The aim of this review is to summarize these biological properties and the potential clinical development of NGF. PMID:26411962

  13. Skeletal effects of growth hormone and insulin-like growth factor-I therapy.

    PubMed

    Lindsey, Richard C; Mohan, Subburaman

    2016-09-01

    The growth hormone/insulin-like growth factor (GH/IGF) axis is critically important for the regulation of bone formation, and deficiencies in this system have been shown to contribute to the development of osteoporosis and other diseases of low bone mass. The GH/IGF axis is regulated by a complex set of hormonal and local factors which can act to regulate this system at the level of the ligands, receptors, IGF binding proteins (IGFBPs), or IGFBP proteases. A combination of in vitro studies, transgenic animal models, and clinical human investigations has provided ample evidence of the importance of the endocrine and local actions of both GH and IGF-I, the two major components of the GH/IGF axis, in skeletal growth and maintenance. GH- and IGF-based therapies provide a useful avenue of approach for the prevention and treatment of diseases such as osteoporosis.

  14. Cytokines and growth factors cross-link heparan sulfate

    PubMed Central

    Migliorini, Elisa; Thakar, Dhruv; Kühnle, Jens; Sadir, Rabia; Dyer, Douglas P.; Li, Yong; Sun, Changye; Volkman, Brian F.; Handel, Tracy M.; Coche-Guerente, Liliane; Fernig, David G.; Lortat-Jacob, Hugues; Richter, Ralf P.

    2015-01-01

    The glycosaminoglycan heparan sulfate (HS), present at the surface of most cells and ubiquitous in extracellular matrix, binds many soluble extracellular signalling molecules such as chemokines and growth factors, and regulates their transport and effector functions. It is, however, unknown whether upon binding HS these proteins can affect the long-range structure of HS. To test this idea, we interrogated a supramolecular model system, in which HS chains grafted to streptavidin-functionalized oligoethylene glycol monolayers or supported lipid bilayers mimic the HS-rich pericellular or extracellular matrix, with the biophysical techniques quartz crystal microbalance (QCM-D) and fluorescence recovery after photobleaching (FRAP). We were able to control and characterize the supramolecular presentation of HS chains—their local density, orientation, conformation and lateral mobility—and their interaction with proteins. The chemokine CXCL12α (or SDF-1α) rigidified the HS film, and this effect was due to protein-mediated cross-linking of HS chains. Complementary measurements with CXCL12α mutants and the CXCL12γ isoform provided insight into the molecular mechanism underlying cross-linking. Fibroblast growth factor 2 (FGF-2), which has three HS binding sites, was also found to cross-link HS, but FGF-9, which has just one binding site, did not. Based on these data, we propose that the ability to cross-link HS is a generic feature of many cytokines and growth factors, which depends on the architecture of their HS binding sites. The ability to change matrix organization and physico-chemical properties (e.g. permeability and rigidification) implies that the functions of cytokines and growth factors may not simply be confined to the activation of cognate cellular receptors. PMID:26269427

  15. Fibrochondrogenesis of hESCs: growth factor combinations and cocultures.

    PubMed

    Hoben, Gwendolyn M; Willard, Vincent P; Athanasiou, Kyriacos A

    2009-03-01

    The successful differentiation of human embryonic stem cells (hESCs) to fibrochondrocyte-like cells and characterization of these differentiated cells is a critical step toward tissue engineering of musculoskeletal fibrocartilages (e.g., knee meniscus, temporomandibular joint disc, and intervertebral disc). In this study, growth factors and primary cell cocultures were applied to hESC embryoid bodies (EBs) for 3 weeks and evaluated for their effect on the synthesis of critical fibrocartilage matrix components: glycosaminoglycans (GAG) and collagens (types I, II, and VI). Changes in surface markers (CD105, CD44, SSEA, PDGFR alpha) after the differentiation treatments were also analyzed. The study was conducted in three phases: (1) examination of growth factors (TGF-beta 3, BMP-2, BMP-4, BMP-6, PDGF-BB, sonic hedgehog protein); (2) comparison of two cocultures (primary chondrocytes or fibrochondrocytes); and (3) the combination of the most effective growth factor and coculture regimen. TGF-beta 3 with BMP-4 yielded EBs positive for collagens I, II, and VI, with up to 6.7- and 4.8-fold increases in GAG and collagen, respectively. Analysis of cell surface markers showed a significant increase in CD44 with the TGF-beta 3 + BMP-4 treatment compared to the controls. Coculture with fibrochondrocytes resulted in up to a 9.8-fold increase in collagen II production. The combination of the growth factors BMP-4 + TGF-beta 3 with the fibrochondrocyte coculture led to an increase in cell proliferation and GAG production compared to either treatment alone. This study determined two powerful treatments for inducing fibrocartilaginous differentiation of hESCs and provides a foundation for using flow cytometry to purify these differentiated cells. PMID:18454697

  16. Regulation of GM-CSF and IL-3 production from the murine keratinocyte cell line PAM 212 following exposure to ultraviolet radiation

    SciTech Connect

    Gallo, R.L.; Staszewski, R.; Sauder, D.N.; Knisely, T.L.; Granstein, R.D. )

    1991-08-01

    Ultraviolet radiation (UVR) exposure induces profound changes in the synthesis and secretion of various cytokines both in vivo and in vitro. Little is known regarding the mechanism of these responses. This investigation evaluated the effects of UVR on the ability of a murine keratinocyte line (PAM 212) to produce interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF). Subconfluent rapidly dividing PAM 212 cells were shown by RNA slot-blot hybridization studies to have increased levels of mRNA for both IL-3 and GM-CSF within 1 h of UVR exposure. However, only GM-CSF-specific bioactivity, as determined by antibody neutralization studies, was shown to increase above baseline in cell supernatants. Cells grown to confluence responded differently to UVR. Under these culture conditions an apparent decrease in bioactivity was detected after UVR exposure for both growth factors, and no change in mRNA levels was detected. In addition to culture density, removal of extracellular calcium or sodium during irradiation, treatment with amiloride, or inhibition of new mRNA synthesis with cordycepin was shown to influence the UVR-induced alteration in release of IL-3 or GM-CSF bioactivity from both confluent and subconfluent PAM 212 cells. These results demonstrate that UVR influences the release of the colony stimulating factors GM-CSF and IL-3 from keratinocyte, and suggests that the state of cell growth and conditions of membrane ion transport influence the mechanisms regulating secretion of those factors.

  17. Fibroblast growth factor (FGF) signaling regulates transforming growth factor beta (TGFβ)-dependent smooth muscle cell phenotype modulation.

    PubMed

    Chen, Pei-Yu; Qin, Lingfeng; Li, Guangxin; Tellides, George; Simons, Michael

    2016-01-01

    Smooth muscle cells (SMCs) in normal blood vessels exist in a highly differentiate state characterized by expression of SMC-specific contractile proteins ("contractile phenotype"). Following blood vessel injury in vivo or when cultured in vitro in the presence of multiple growth factors, SMC undergo a phenotype switch characterized by the loss of contractile markers and appearance of expression of non-muscle proteins ("proliferative phenotype"). While a number of factors have been reported to modulate this process, its regulation remains uncertain. Here we show that induction of SMC FGF signaling inhibits TGFβ signaling and converts contractile SMCs to the proliferative phenotype. Conversely, inhibition of SMC FGF signaling induces TGFβ signaling converting proliferating SMCs to the contractile phenotype, even in the presence of various growth factors in vitro or vascular injury in vivo. The importance of this signaling cross-talk is supported by in vivo data that show that an SMC deletion of a pan-FGF receptor adaptor Frs2α (fibroblast growth factor receptor substrate 2 alpha) in mice profoundly reduces neointima formation and vascular remodelling following carotid artery ligation. These results demonstrate that FGF-TGFβ signaling antagonism is the primary regulator of the SMC phenotype switch. Manipulation of this cross-talk may be an effective strategy for treatment of SMC-proliferation related diseases. PMID:27634335

  18. Fibroblast growth factor (FGF) signaling regulates transforming growth factor beta (TGFβ)-dependent smooth muscle cell phenotype modulation

    PubMed Central

    Chen, Pei-Yu; Qin, Lingfeng; Li, Guangxin; Tellides, George; Simons, Michael

    2016-01-01

    Smooth muscle cells (SMCs) in normal blood vessels exist in a highly differentiate state characterized by expression of SMC-specific contractile proteins (“contractile phenotype”). Following blood vessel injury in vivo or when cultured in vitro in the presence of multiple growth factors, SMC undergo a phenotype switch characterized by the loss of contractile markers and appearance of expression of non-muscle proteins (“proliferative phenotype”). While a number of factors have been reported to modulate this process, its regulation remains uncertain. Here we show that induction of SMC FGF signaling inhibits TGFβ signaling and converts contractile SMCs to the proliferative phenotype. Conversely, inhibition of SMC FGF signaling induces TGFβ signaling converting proliferating SMCs to the contractile phenotype, even in the presence of various growth factors in vitro or vascular injury in vivo. The importance of this signaling cross-talk is supported by in vivo data that show that an SMC deletion of a pan-FGF receptor adaptor Frs2α (fibroblast growth factor receptor substrate 2 alpha) in mice profoundly reduces neointima formation and vascular remodelling following carotid artery ligation. These results demonstrate that FGF-TGFβ signaling antagonism is the primary regulator of the SMC phenotype switch. Manipulation of this cross-talk may be an effective strategy for treatment of SMC-proliferation related diseases. PMID:27634335

  19. Synergistic Action of Fibroblast Growth Factor-2 and Transforming Growth Factor-beta1 Enhances Bioprinted Human Neocartilage Formation

    PubMed Central

    Cui, Xiaofeng; Breitenkamp, Kurt; Lotz, Martin; D’Lima, Darryl

    2012-01-01

    Bioprinting as a promising but unexplored approach for cartilage tissue engineering has the advantages of high throughput, digital control, and highly accurate placement of cells and biomaterial scaffold to the targeted 3D locations with simultaneous polymerization. This study tested feasibility of using bioprinting for cartilage engineering and examined the influence of cell density, growth and differentiation factors. Human articular chondrocytes were printed at various densities, stimulated transiently with growth factors and subsequently with chondrogenic factors. Samples were cultured for up to 4 weeks to evaluate cell proliferation and viability, mechanical properties, mass swelling ratio, water content, gene expression, ECM production, DNA content, and histology. Bioprinted samples treated with FGF-2/TGF-β1 had the best chondrogenic properties among all groups apparently due to synergistic stimulation of cell proliferation and chondrogenic phenotype. ECM production per chondrocyte in low cell density was much higher than that in high cell seeding density. This finding was also verified by mechanical testing and histology. In conclusion, cell seeding density that is feasible for bioprinting also appears optimal for human neocartilage formation when combined with appropriate growth and differentiation factors. PMID:22508498

  20. Measuring growth hormone and insulin-like growth factor-I in infants: what is normal?

    PubMed

    Hawkes, Colin Patrick; Grimberg, Adda

    2013-12-01

    The role of growth hormone (GH) and insulinlike growth factor-I (IGF-I) change through early childhood. Whereas poor growth is a later presenting feature, infants with isolated GH deficiency have a normal birth weight and length, and often present with hypoglycemia. IGF-I plays an important role antenatally and post-natally in somatic and brain growth. In order to evaluate the GH/IGF-I axis in infancy, an understanding of the normal physiology is required. Measurements of GH and IGF-I in this population should be interpreted in the context of the assays used, as well as their limitations. In this review, we summarize our current understanding of normal GH and IGF-I secretion in children under 18 months of age, and describe variations in the reported assay-specific measurements.

  1. Measuring Growth Hormone and Insulin-like Growth Factor-I in Infants: What is Normal?

    PubMed Central

    Hawkes, Colin Patrick; Grimberg, Adda

    2014-01-01

    The role of growth hormone (GH) and insulin-like growth factor-I (IGF-I) change through early childhood. Whereas poor growth is a later presenting feature, infants with isolated GH deficiency have a normal birth weight and length, and often present with hypoglycemia. IGF-I plays an important role antenatally and post-natally in somatic and brain growth. In order to evaluate the GH/IGF-I axis in infancy, an understanding of the normal physiology is required. Measurements of GH and IGF-I in this population should be interpreted in the context of the assays used, as well as their limitations. In this review, we summarize our current understanding of normal GH and IGF-I secretion in children under 18 months of age, and describe variations in the reported assay-specific measurements. PMID:24575549

  2. Epidermal growth factor receptor inhibitor therapy for recurrent respiratory papillomatosis

    PubMed Central

    Sidman, James D.

    2013-01-01

    The epidermal growth factor pathway has been implicated in various tumors, including human papillomavirus (HPV) lesions such as recurrent respiratory papillomatosis (RRP). Due to the presence of epidermal growth factor receptors in RRP, epidermal growth factor receptor (EGFR) inhibitors have been utilized as adjuvant therapy. This case series examines the response to EGFR inhibitors in RRP. Four patients with life-threatening RRP were treated with EGFR inhibitors. Operative frequency and anatomical Derkay scores were calculated prior to, and following EGFR inhibitor treatment via retrospective chart review. The anatomical Derkay score decreased for all four patients after initiation of EGFR inhibitor therapy. In one patient, the operative frequency increased after switching to an intravenous inhibitor after loss of control with an oral inhibitor. In the other patients there was a greater than 20% decrease in operative frequency in one and a more than doubling in the time between procedures in two.  This study suggests that EGFR inhibitors are a potential adjuvant therapy in RRP and deserve further study in a larger number of patients. PMID:24795806

  3. In vivo cartilage formation from growth factor modulated articular chondrocytes.

    PubMed

    Bradham, D M; Horton, W E

    1998-07-01

    Recent procedures for autologous repair of cartilage defects may be difficult in elderly patients because of the loss of stem cells and chondrocytes that occurs with age and the slow in vitro proliferation of chondrocytes from aged cartilage. In this study secondary chondroprogenitor cells were obtained by modulating the phenotype of articular chondrocytes with growth factors and stimulating the proliferation of these cells in culture. Chondrocytes isolated from the articular cartilage of mature New Zealand White rabbits were exposed to a combination of transforming growth factor beta and basic fibroblast growth factor treatment. These cells ceased the production of Collagen II (a marker for the chondrocyte phenotype) and underwent a 136-fold increase in cell number. Next, the cells were placed in high density culture and reexpressed the chondrocyte phenotype in vitro and formed hyaline cartilage in an in vivo assay. Primary chondrocytes obtained from articular cartilage of elderly humans could be manipulated in a similar fashion in vitro. These human secondary chondroprogenitor cells formed only cartilage tissue when assayed in vivo and in tissue bioreactors. This approach may be essential for autologous repair of degenerated articular cartilage in elderly patients with osteoarthritis.

  4. Modulation of endothelin-1 in normal human keratinocytes by UVA1/B radiations, prostaglandin E2 and peptidase inhibitors.

    PubMed

    Pernet, I; Mayoux, C; Trompezinski, S; Schmitt, D; Viac, J

    2000-12-01

    In the skin, keratinocytes synthesize and secrete endothelin-(ET-1), a potent vasoconstrictor peptide which acts also as a growth factor for most skin cells. The aim of the study was to test the effects of UVA1 and the associations UVA1/B on the expression of ET-1 in normal human keratinocytes and to determine whether exogenously added prostaglandin E2 (PGE2) regulated ET-1 expression. As ET-1 is susceptible to degradation, we also evaluated whether ET-1 secretion was modulated by peptidase inhibitors. Our results showed that UVA1 (365 nm) did not modify the levels of preproET-1 mRNA and protein. Moreover, the associations UVA1+UVB or UVB+UVA1 down-regulated the overexpression of secreted ET-1 induced by UVB alone. PGE2 at 10(-5) M reduced the expression of ET-1 at the mRNA and protein levels but did not exert any significant modification at lower concentrations from 10(-10) to 10(6) M. Phosphoramidon, an endothelin converting enzyme (ECE) inhibitor, drastically decreased the amount of ET-1 accumulating in the culture medium in basal conditions or after UVB irradiation. Conversely, thiorphan, a specific inhibitor of neutral endopeptidase (NEP), rather increased the levels of ET-1 secretion mainly after UVB irradiation. Taken together, the results showed that normal human keratinocytes secrete and partly degrade ET-1 through ECE and NEP pathways and pointed out a differential regulation of ET-1 by UVB and UVA1 radiations without any noticeable role for PGE2.

  5. Vascular endothelial growth factor, platelet-derived endothelial cell growth factor and angiogenesis in non-small-cell lung cancer

    PubMed Central

    O'Byrne, K J; Koukourakis, M I; Giatromanolaki, A; Cox, G; Turley, H; Steward, W P; Gatter, K; Harris, A L

    2000-01-01

    High microvessel density, an indirect measure of angiogenesis, has been shown to correlate with increased tumour size, lymph node involvement and poor prognosis in non-small-cell lung cancer (NSCLC). Tumour cell vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) expression correlate with angiogenesis and a poor outcome in this disease. In a retrospective study VEGF and PD-ECGF expression and microvessel density were evaluated immunohistochemically in surgically resected specimens (T1–3, N0–2) from 223 patients with operable NSCLC using the VG1, P-GF.44C and JC70 monoclonal antibodies respectively. High VEGF immunoreactivity was seen in 104 (46.6%) and PD-ECGF in 72 (32.3%) cases and both were associated with high vascular grade tumours (P = 0.009 and P = 0.05 respectively). Linear regression analysis revealed a weak positive correlation between VEGF and PD-ECGF expression in cancer cells (r = 0.21;P = 0.002). Co-expression of VEGF and PD-ECGF was not associated with a higher microvessel density than VEGF or PD-ECGF only expressing tumours. Furthermore a proportion of high vascular grade tumours expressed neither growth factor. Univariate analysis revealed tumour size, nodal status, microvessel density and VEGF and PD-ECGF expression as significant prognostic factors. Tumour size (P< 0.02) and microvessel density (P< 0.04) remained significant on multivariate analysis. In conclusion, VEGF and PD-ECGF are important angiogenic growth factors and have prognostic significance in NSCLC. Furthermore the study underlines the prognostic significance of microvessel density in operable NSCLC. © 2000 Cancer Research Campaign PMID:10780522

  6. Effects of low frequency pulsed electrical current on keratinocytes in vitro

    SciTech Connect

    Hinsenkamp, M.; Jercinovic, A.

    1997-05-01

    The effects of low frequency pulsed electrical current on epidermal repair in vitro were examined. Charge-balanced current stimuli proposed for chronic wound treatment were tested on skin keratinocytes cultured at an air-liquid interface on dead human dermis. Results imply that the balance between proliferation and differentiation in electrically treated samples is significantly modified in favor of differentiation. More advanced differentiation, shown through epidermal histology, was obtained in cultures exposed to electrical current, whereas the culture growth, the result of keratinocyte migration and proliferation, was greater in control samples.

  7. Human Papilloma Viral DNA Replicates as a Stable Episome in Cultured Epidermal Keratinocytes

    NASA Astrophysics Data System (ADS)

    Laporta, Robert F.; Taichman, Lorne B.

    1982-06-01

    Human papilloma virus (HPV) is poorly understood because systems for its growth in tissue culture have not been developed. We report here that cultured human epidermal keratinocytes could be infected with HPV from plantar warts and that the viral DNA persisted and replicated as a stable episome. There were 50-200 copies of viral DNA per cell and there was no evidence to indicate integration of viral DNA into the cellular genome. There was also no evidence to suggest that viral DNA underwent productive replication. We conclude that cultured human epidermal keratinocytes may be a model for the study of certain aspects of HPV biology.

  8. KLF10, transforming growth factor-{beta}-inducible early gene 1, acts as a tumor suppressor

    SciTech Connect

    Song, Ki-Duk; Kim, Duk-Jung; Lee, Jong Eun; Yun, Cheol-Heui; Lee, Woon Kyu

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer KLF10{sup -/-} mice exhibited accelerated papilloma development after DMBA/TPA treatment. Black-Right-Pointing-Pointer KLF10{sup -/-} keratinocytes showed increased proliferation and apoptosis. Black-Right-Pointing-Pointer KLF10{sup -/-} MEFs yielded more colonies than wild-type one with H-Ras transfection. Black-Right-Pointing-Pointer KLF10 dose-dependently activated p21{sup WAF1/CIP1} transcription. Black-Right-Pointing-Pointer KLF10 is a tumor suppressor and that it targets p21{sup WAF1/CIP1} transcription. -- Abstract: Krueppel-like factor 10 (KLF10) has been suggested to be a putative tumor suppressor. In the present study, we generated KLF10 deficient mice to explore this hypothesis in vivo. KLF10 deficient mice exhibited increased predisposition to skin tumorigenesis and markedly accelerated papilloma development after DMBA/TPA treatment. On the other hand, KLF10 deficient keratinocytes showed increased proliferation and apoptosis. In colony formation assays after oncogenic H-Ras transfection, KLF10 deficient mouse embryonic fibroblasts (MEFs) yielded more colonies than wild-type MEFs. Furthermore, KLF10 dose-dependently activated p21{sup WAF1/CIP1} transcription, which was independent of p53 and Sp1 binding sites in p21{sup WAF1/CIP1} promoter. This study demonstrates that KLF10 is a tumor suppressor and that it targets p21{sup WAF1/CIP1} transcription.

  9. Semiquantitative reverse transcription-polymerase chain reaction analysis of mRNA for growth factors and growth factor receptors from normal and healing rabbit medial collateral ligament tissue.

    PubMed

    Sciore, P; Boykiw, R; Hart, D A

    1998-07-01

    Growth factors and their receptors play an essential role in the development, maturation, and response to injury of all tissues. A number of studies have explored the possibility of improving ligament healing with exogenous growth factors. However, limited data is available regarding the endogenous growth factor network in ligaments on which any exogenous growth