OBJECTIVE

To evaluate the efficacy of cultured cutaneous substitute (CCS) in accelerating the healing of combined radiation-skin wound injury (CRWI) in minipigs.

METHODS

Autologous porcine bone marrow-derived mesenchymal stem cells (BMSC) and skin-derived keratinocytes (SK) were infected by recombinant retrovirus expressing human (h) platelet-derived growth factor-A (hPDGF-A). CCS was constructed by loading acellular human amniotic membrane (HAM) with normal porcine BMSC and SK (BMSC-/SK-CCS) or with hPDGF-A modified counterparts (BMSC+/SK+CCS). The expression of exogenous hPDGF-A in cells and CCS was assessed by reverse transcription polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA). The CCS or HAM were grafted to the dorsal CRWI sites (20 Gy local irradiation plus full-thickness skin removal, diameter = 40 mm) of minipigs. Wound healing rate and pathological changes were observed.

RESULTS

High levels of hPDGF-A expression were confirmed in gene-modified cells (3780 pg/ml), cultured CCS (506 pg/ml) and transplanted CCS (250 pg/ml). The transplantation of the BMSC+/SK+CCS resulted in a shorter healing time (16-18, days) (P < 0.05 vs. other groups). The healing rates ranked as BMSC+/SK+CCS>> BMSC-/SK-CCS>> HAM>> wound control. Pathologically, there were better granulation formation and re-epithelialisation, and collagen deposition in BMSC+/SK+CCS-treated wound than those in other groups. The angiogenesis ability followed the same order as healing rate of different groups. At day 7, the area densities of vasculature in granulation tissue of group BMSC+/SK+CCS, BMSC-/SK-CCS, HAM, wound only were 15.4, 10.3, 6.0 and 5.7%, respectively, while the number densities of vasculature was 767, 691, 126 and 109 (number/mm(2)), respectively.

CONCLUSIONS

Topical transplantation of hPDGF-A modified CCS may be applicable to the management of refractory wounds.

Progress in biotechnology has led to new therapeutic approaches in various fields of human health care, such as the autologous grafting of cultured epidermal cell sheets on burned patients. These cultures depend on various parameters but <em>growth</em> <em>factors</em> are of paramount importance. Cutaneous cells are known to secrete various <em>growth</em> <em>factors</em> in vivo, although only a few have been identified. The aim of this study was to determine if such <em>factors</em> are secreted from human cutaneous cells in culture, to evaluate their effects on epidermal cell proliferation in vitro and to analyse them on SDS-PAGE. Human skin fibroblasts and <em>keratinocytes</em> were co-cultured for 8-10 days using a Costar trans-filter system. Dermo-epidermal cooperation was observed in such a co-culture system through the exchange of secretion products in the culture medium. Epidermal cell <em>growth</em> and metabolic activities were highly stimulated in co-culture (<em>2</em>-fold and 1.5-fold, respectively, P < 0.0<em>2</em>) compared to the control. The de novo synthesis of secretion products, notably of a protein of about 40 kDa, was specifically induced in co-culture. The identification of new <em>keratinocyte</em> <em>growth</em> <em>factors</em> could accelerate graftable epidermal sheet production in vitro for human wound coverage and possibly enhance wound healing in vivo.

BACKGROUND

Both psoralen plus ultraviolet (UV) A (PUVA) and narrowband UVB (NB-UVB) irradiation are effective treatments for vitiligo vulgaris. However, the mechanisms of PUVA and NB-UVB in repigmentation are not thoroughly clarified. Our previous results showed that NB-UVB irradiation directly promotes melanocyte (MC) migration and stimulates MC proliferation via keratinocytes (KCs).

OBJECTIVE

In the present study, we used NB-UVB as a reference for comparison to investigate the immediate effects of PUVA on MC proliferation and migration.

METHODS

Cultured MCs and KCs were treated with PUVA or irradiated with NB-UVB. The direct impact of PUVA treatment on MCs was assessed in terms of its effect on MC proliferation and migration. The indirect effect of PUVA treatment and NB-UVB irradiation on MC proliferation via KCs was also investigated. The activities of matrix metalloproteinase (MMP)-2 and MMP-9, known for their influence on cell migration, were evaluated in the PUVA-treated MC and KC supernatants. The concentrations of MC mitogens/growth factors in the PUVA-treated KC supernatants were also determined. In addition, the serum levels of MC mitogens/growth factors in healthy controls, in patients with active vitiligo and in patients with repigmenting vitiligo after PUVA treatment were determined to elucidate the mechanisms of how PUVA induces vitiligo repigmentation in vivo.

RESULTS

Our results demonstrated that PUVA treatment did not significantly stimulate the release of MC mitogens/growth factors from KCs. The migration of MCs was also not enhanced after PUVA treatment. The expression of MMP-2 activity in supernatants derived from PUVA-treated MCs was significantly increased as compared with the control group. However, neither MMP-2 nor MMP-9 activity in KC supernatants was stimulated by PUVA treatment. In contrast to NB-UVB, immediate effects of PUVA on MC proliferation and migration were not observed in this study. Sera from patients with repigmenting vitiligo after PUVA treatment contained higher levels of basic fibroblast growth factor, stem cell factor and hepatocyte growth factor as compared with healthy controls and patients with active vitiligo.

CONCLUSIONS

Our results indicate that in addition to immune suppression, PUVA treatment creates a favourable milieu for promoting the growth of MCs in patients with vitiligo instead of directly stimulating the regrowth of MCs. Based on our results, we propose that in the active stage of vitiligo, PUVA treatment is the therapy of choice to slow down the destruction of MCs and to create a favourable environment for MCs to survive. In the stable stage of vitiligo, NB-UVB irradiation should be used to stimulate the proliferation and migration of MCs directly.

Similar cellular responses are elicited by retinoic acid (RA) and transforming <em>growth</em> <em>factor</em> beta (TGF-beta). We investigated the ability of RA to modulate the production of TGF-beta in normal human <em>keratinocytes</em> (HKc) and HKc lines immortalized by transfection with human papillomavirus type 16 DNA (HKc/HPV16). RA treatment of both normal HKc and HKc/HPV16 resulted in a <em>2</em>-3-fold induction in secreted levels of latent TGF-beta. The induction in TGF-beta secretion by RA was dose dependent, with significant increases observed with RA concentrations as low as 1-10 nM, and time dependent, with maximal induction occurring about 3 days after initiation of RA exposure. In addition, RA induced intracellular levels of TGF-beta almost 5-fold. Sandwich enzyme-linked immunosorbent assays were used to specifically quantify TGF-beta 1 and TGF-beta <em>2</em> secreted by normal HKc and HKc/HPV16 cultured in the absence or presence of RA. RA increased the secreted levels of latent TGF-beta 1 and TGF-beta <em>2</em> an average of <em>2</em>- and 5-fold, respectively, with no major differences in the fold induction between normal HKc and HKc/HPV16. Northern blot analysis of mRNA isolated from HKc/HPV16 demonstrated that RA treatment induced specific transcripts for TGF-beta 1 and TGF-beta <em>2</em> about 3- and 50-fold, respectively. RA treatment of HKc had no significant effect on the binding affinity of TGF-beta for its receptors or receptor number. Normal HKc and HKc/HPV16 displayed similar dose-dependent inhibition of proliferation by TGF-beta 1. These studies indicate that RA may regulate <em>growth</em> control in both normal HKc and HKc/HPV16 by enhancing TGF-beta 1 and TGF-beta <em>2</em> production, which, after activation at the cell surface, could inhibit cellular proliferation in an autocrine and/or paracrine manner.

The suppressor of cytokine signaling (SOCS)-3 has been shown to impair proliferation and migration of <em>keratinocytes</em>. To assess the functional dependency among wound inflammation, SOCS-3 induction in <em>keratinocytes</em>, and the outcome of healing, we generated a transgenic mouse that specifically overexpresses SOCS-3 in <em>keratinocytes</em>. Acute wound healing in transgenic mice was severely impaired. <em>Keratinocyte</em>-specific overexpression of SOCS-3 led to atrophied wound-margin epithelia and augmented the inflammatory response of wound <em>keratinocytes</em> by an increase in chemokine (MIP-<em>2</em>) and inflammatory enzyme (COX-<em>2</em> and iNOS) expression. In addition, wound tissue of transgenic mice showed a prolonged persistence of neutrophils and macrophages. Remarkably, impaired wounds showed elevated levels of transforming <em>growth</em> <em>factor</em> (TGF)-beta1, which appeared to interfere with healing, as its neutralization markedly improved wound closure in transgenic mice. Interestingly, administration of a TGF-beta-neutralizing antibody increased wound inflammation in nontransgenic mice but not in transgenic littermates. This study suggests that SOCS-3-driven disturbances in wound <em>keratinocytes</em> are sufficient to induce inflamed wound conditions that resemble characteristics of chronic wounds in mice.

BACKGROUND

Tissue <em>factor</em> (TF) and <em>factor</em> (F) VIIa are the primary initiators of the coagulation cascade, but also promote non-hemostatic events, such as angiogenesis and tumor <em>growth</em>, via activation of protease activated receptor-<em>2</em> (PAR<em>2</em>). Our previous findings indicated that the TF:FVIIa complex activates signal transducer and activator of transcription (STAT) signaling, leading to cell survival in TF-transfected baby hamster kidney (BHK) cells.

METHODS

Using BHK TF, keratinocytes (HaCaT) and human umbilical vein endothelial cells (HUVEC), FVIIa-induced phosphorylation and activation of the transcription factor cyclic AMP-responsive binding protein (CREB) were tested and compared to that elicited by thrombin and FXa. In addition, the effect of these factors on cell survival and expression of apoptosis-associated proteins was monitored.

RESULTS

Factor VIIa led to a TF-dependent, but TF cytoplasmic domain-independent phosphorylation and activation of CREB in BHK TF, HaCaT and HUVEC. CREB activation was sensitive to blockade of the extracellular-signal regulated kinase 1/<em>2</em> pathway and PAR<em>2</em>. Surprisingly, FVIIa decreased cell survival in HaCaT cells but not other cell types and upregulated the pro-apoptotic proteins Bak and Puma in a CREB-dependent manner. Factor Xa, but not FIIa, induced phosphorylation of CREB, but did not have an effect on apoptosis.

CONCLUSIONS

TF:FVIIa induces CREB phosphorylation and activation in several cell types, but TF:FVIIa induces pro-apoptotic proteins and apoptosis only in selected cell types.

The three mammalian isoforms of transforming <em>growth</em> <em>factor</em>-beta(TGF-beta1, beta<em>2</em>, beta3) are potent regulators of cell <em>growth</em>, differentiation, and extracellular matrix deposition. To study their role in skin differentiation, we investigated the expression of TGF-beta isoforms on cell <em>growth</em> and differentiation induction of the human <em>keratinocyte</em> cell line, HaCaT by elevating the Ca(<em>2</em>+) concentration. An ELISA and RT-PCR assay revealed secreted TGF-beta 1 protein and TGF-beta 1 mRNA were increased during calcium-induced differentiation. In contrast, major differences were seen for TGF-beta <em>2</em> and TGF-beta 3 mRNA which were decreased during differentiation, but TGF-beta <em>2</em> and TGF-beta3 protein were not evident on an ELISA. These results suggest different functions for each TGF-beta isoforms in epidermal differentiation, such that TGF-beta 1 is associated with the more differentiated state, and TGF-beta <em>2</em> and TGF-beta 3 may be associated the more proliferated state.

Heparin-binding EGF-like <em>growth</em> <em>factor</em> (HB-EGF) and amphiregulin (AREG) are the members of EGF family that bind to common EGF receptor (EGFR) in the epidermis. However, the role of these two <em>growth</em> <em>factors</em> in epidermal hyperplasia of psoriasis has not been established. On the other hand, CD4+ T cells are responsible for the development of the psoriatic plaques. However, inflammatory cytokines, such as TNFalpha, IL-1beta and IFNgamma, inhibit the <em>growth</em> of human <em>keratinocytes</em> in vitro. The expression of HB-EGF, AREG and EGFR proteins in normal (n = <em>2</em><em>2</em>) and psoriatic (n = 34) skin tissues was examined by immunohistochemistry. Then, the effects of HB-EGF and AREG on the <em>growth</em> of cultured adult normal human epidermal <em>keratinocytes</em> (NHEK-AD) with or without TH1 cytokines, such as TNFalpha, IL-1beta and IFNgamma, were examined by 3-(4,5-dimethyl-<em>2</em>-thiazolyl)-<em>2</em>,5-diphenyl-<em>2</em>H-tetrazolium bromide (MTT) assay, and the effects of these cytokines on the expression of EGFR mRNA in NHEK-AD were examined by real-time reverse transcriptase-polymerase chain reaction. The expression of HB-EGF and AREG in the epidermis was not specific to psoriatic plaques, but the distribution of positive cells throughout the epidermis was different between normal skins and psoriatic plaques. On the other hand, in the dermis and the papillary dermis, most of vascular endothelial cells and infiltrating mononuclear cells expressed both HB-EGF and AREG in normal skins and psoriatic plaques, and these positive cells were more frequent in psoriasis compared to normal skin. In the in vitro <em>growth</em> assay, HB-EGF, not AREG, stimulated the proliferation of NHEK-AD at the optimal concentration of 1 ng/ml. Furthermore, HB-EGF compensated the <em>growth</em>-suppressing effects of TNFalpha, IL-1beta and IFNgamma on NHEK-AD, and TNFalpha promoted the <em>growth</em> of NHEK-AD at the concentration of <em>2</em> and <em>2</em>0 U/ml in combination with HB-EGF and, in lesser extent, with AREG. However, TNFalpha did not affect the expression of EGFR mRNA in NHEK-AD. <em>Growth</em> <em>factors</em> and inflammatory cytokines produced in the dermis would be important for the epidermal proliferation in psoriatic plaques and TNFalpha may play a key role in cooperation with HB-EGF and AREG in the proliferation of epidermal <em>keratinocytes</em> at the psoriatic skin lesions.

Cell-to-cell interactions between melanocytes and <em>keratinocytes</em> increase the proliferation and migration of melanocytes. In fact, mixed <em>keratinocyte</em> 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 <em>keratinocytes</em> 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 <em>keratinocytes</em>. This may be related to increases in stem cell <em>factor</em> and basic fibroblast <em>growth</em> <em>factor</em>, <em>growth</em> <em>factors</em> for melanocytes, produced by the ADSCs. The ratios of melanocytes stained with antibodies against Trp-<em>2</em>, E-cadherin and N-cadherin were significantly increased by co-culturing with ADSCs compared with co-culturing with <em>keratinocytes</em> 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.

There is some evidence that proliferation of pleural mesothelial cells (MC) occurs soon after deposition of asbestos fibers. To study this effect, we instilled a single dose of 0.1 mg crocidolite into the lungs of mice for 1 and 6 weeks and counted labeled nuclei after 3H-thymidine (3HT) injection. Short fibers (< 1 micron) induced little change in the lung; they were mostly phagocytized and had a minimal effect on MC labeling. Long fibers up to <em>2</em>0 microns damaged the bronchiolar epithelium and were incorporated into connective tissue. Increased 3HT uptake was seen in fibroblasts and epithelial cells and also in MC, which peaked at <em>2</em>% labeled at 1 week compared to near 0% labeling in controls. No fibers were found in or near labeled MC, which suggested that a cytokine generated in the lung during the early response phase might induce MC proliferation. To look for a cytokine effect in vitro, we instilled asbestos into rat lungs and, after 1 and 6 weeks, bronchoalveolar and pleural lavage fluids as well as macrophages were collected. Alveolar macrophages contained fibers, but pleural macrophages (PM) did not. After short-term culture, macrophage supernatants and the lavage fluids were tested on rat lung MC in culture. At 1 week, PM secreted <em>growth</em> <em>factor</em>(s) for MC, and the mitogenic effect was more pronounced with lavage fluids. No effects on MC were found using material prepared 6 weeks after asbestos. The early MC <em>growth</em> increase was not blocked by antibodies to cytokines, such as platelet-derived <em>growth</em> <em>factor</em>, fibroblast <em>growth</em> <em>factors</em>, or tumor necrosis <em>factor</em>, but was inhibited by anti-<em>keratinocyte</em> <em>growth</em> <em>factor</em> (anti-KGF). The results show that an early <em>growth</em> phase of MC after asbestos exposure appears unrelated to particle translocation to the pleura but is associated with cytokine release, most likely KGF, by lung cells.

OBJECTIVE

Repifermin (<em>keratinocyte</em> <em>growth</em> <em>factor</em>-<em>2</em>, KGF-<em>2</em>) is a <em>growth</em> <em>factor</em> that selectively induces epithelial cell proliferation, differentiation and migration. The objective of this study was to assess the effect of repifermin on in vitro tumor cell proliferation and in vivo tumor <em>growth</em> using a variety of human carcinoma cell lines with differing <em>growth</em> rates and levels of KGF receptor (KGFR) expression.

METHODS

Potential effects of repifermin on in vitro cell proliferation were evaluated by alamarBlue and/or [(3)H]-thymidine incorporation assays under a range of serum conditions. In vivo tumor growth was evaluated by implanting KGFR(+) carcinomas subcutaneously into nude mice and measuring tumor growth over time in mice injected intravenously (i.v.) or intraperitoneally (i.p.) with repifermin or placebo.

RESULTS

In vitro, none of the 30 human carcinoma cell lines tested demonstrated a substantial increase in proliferation in response to repifermin over the concentration range 0.01 to 1000 ng/ml. In vivo results showed no significant tumor <em>growth</em>-promoting activity when single- or multiple-cycle intravenous injections of repifermin (1 mg/kg) were given to athymic nude mice inoculated with human KGFR(+) tumors of the pharynx (Detroit 56<em>2</em>, FaDu), colon (Caco-<em>2</em>), salivary gland (A-<em>2</em>53) or tongue (SCC-<em>2</em>5, CAL <em>2</em>7). In addition, repifermin (0.<em>2</em> or <em>2</em> mg/kg) injected i.p. for <em>2</em> weeks had no effect on the <em>growth</em> of eight other human carcinomas including those of the ovary (NIH:OVCAR-3, SK-OV 3, PA-1), bladder (SCaBER), epidermis (A 431), lung (SW 900), breast (MDA-MB-<em>2</em>31) and cervix (SiHa).

CONCLUSIONS

Repifermin had no in vitro or in vivo proliferative effects on KGFR(+) human epithelial-like tumors. This failure to stimulate tumor cell growth highlights the ability of repifermin to specifically target normal epithelial tissue. This is critical to the safety profile of repifermin, since it is currently in phase II clinical trials for the treatment of cancer patients with mucositis resulting from chemo- or radiotherapy.

The ultraviolet B (UVB) portion (<em>2</em>80-3<em>2</em>0 nm) of solar radiation is considered to be a major etiologic <em>factor</em> in human skin cancer and is a known cause of extensive DNA damage. In this study, we observed that UVB exposure of immortalized epidermal <em>keratinocytes</em> (HaCat cells) harboring mutant p53 leads to G(<em>2</em>)/M cell cycle arrest in both asynchronously <em>growing</em> and synchronized cells in a dose dependent manner. Following UVB exposure (<em>2</em>00 mJ/cm(<em>2</em>)), we observed a threefold increase in G(<em>2</em>)/M population at 6 h, which increased to sixfold. The observed G(<em>2</em>)/M arrest was associated with an increase in cyclin B level whereas cdc<em>2</em> protein remained unchanged. However, we observed an accumulation of tyrosine 15 hyperphosphorylated cyclin B-cdc<em>2</em> complex. In addition, we observed an increase in chk1 kinase and a decrease in cdc<em>2</em>5C protein levels. Chk1 phosphorylates cdc<em>2</em>5C on serine <em>2</em>16 and inactivates it whereas cdc<em>2</em>5C dephosphorylates tyrosine 15 phosphate of cdc<em>2</em> and activates the cdc<em>2</em>-cyclin B complex. Therefore, the increase in chk1 and the decrease in cdc<em>2</em>5C both participate in inhibiting the G<em>2</em>/M transition. Our data identifies two upstream targets leading to inhibition of cyclin B-cdc<em>2</em> complexes, which explain the inhibition in cyclin B-associated cdc<em>2</em> kinase following UVB exposure. The inactive phosphorylated cdc<em>2</em>-cyclin B complex remains sequestered in cytoplasm and may migrate to the nucleus following activation. Our data also indicate that UVB exerts unique effects in different types of skin <em>keratinocytes</em> having nonfunctional or mutant p53.

The pathology of chronic dermal ulcers is characterized by excessive proteolytic activity which degrades extracellular matrix (required for cell migration) and <em>growth</em> <em>factors</em> and their receptors. The transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) has been identified as an important component of wound healing. Recent developments in molecular therapy offer exciting prospects for the modulation of wound healing, specifically those targeting TGF-beta. We investigated expression of angiogenic bFGF and VEGF, and collagenases MMP-<em>2</em> and MMP-9 in tissue samples from chronic dermal wounds by immunohistochemistry. The effect of TGF-beta targeting using antisense oligonucleotides on the expression of these <em>factors</em> was analysed by ELISA in human <em>keratinocytes</em>. Immunohistochemical investigation demonstrated a decreased expression of bFGF and VEGF protein, and an increased expression of MMP-<em>2</em> and MMP-9 in tissue samples from chronic dermal wounds compared to normal human skin. Antisense TGF-beta oligonucleotide treatment down-regulated collagenase secretion activity and up-regulated VEGF secretion in vitro. Therefore, TGF-beta antisense oligonucleotide technology may be a potential therapeutic option for the inhibition of proteolytic tissue destruction, and stimulation of angiogenesis in chronic wounds. Improving basic knowledge and pharmaceutical intervention in this area ultimately may help clinicians to identify and proactively intervene in an effort to prevent normal wounds from becoming chronic.

Normal human <em>keratinocytes</em> (KC) are a prominent source of vascular endothelial <em>growth</em> <em>factor</em> (VEGF)/vascular permeability <em>factor</em> (VPF), both in vivo and in tissue culture. In this report we have investigated the influence of retinoids, which are used to treat several skin diseases, on VEGF/VPF production by KC. All-trans retinoic acid (RA), 13-cis RA, and all-trans retinol reduced VEGF/VPF secretion by KC in primary cultures by a mean +/- SD of 58 +/- <em>2</em>5%, 46 +/- <em>2</em>1%, and 54 +/- <em>2</em>0%, respectively, compared with control values. Reductions were observed at concentrations as low as 10(-10)M for all-trans RA, a level that is easily reached in vivo during retinoid treatment. The reduction in VEGF/VPF protein by 10(-6)M all-trans RA was paralleled by a strong downregulation of VEGF/VPF mRNA levels. In contrast to normal KC, all-trans RA had no effect on the HaCaT <em>keratinocyte</em> cell line, and it stimulated VEGF/VPF release by the epidermoid carcinoma cell line A431 <em>2</em>-fold. Our data demonstrate that retinoids are potent inhibitors of VEGF/VPF production by normal human KC. Downregulation of VEGF/VPF production in these cells by retinoids may contribute to the therapeutic effects or retinoids in diseases that are accompanied by angioproliferation, such as psoriasis.

OBJECTIVE

Topical pretreatment with aIl-trans-retinoic acid (atRA) is known to improve healing of cutaneous wounds. We tested the effect of atRA on wound healing of genetically diabetic db/db mice. It is known that cutaneous wounds of db/db mice show delayed wound healing due to impaired wound contraction, delayed granulation tissue formation and underexpression of keratinocyte growth factor (KGF).

METHODS

0.1% atRA in 100 mg aqueous gel was applied to the back skin of db/db mice as well as to their normal heterozygous littermates, db/+ mice, for five consecutive days, and 2 days after completion of the atRA treatment, two round excisional wounds were created down the panniculus carnosus with a 6-mm punch biopsy on the back skin of each mouse.

RESULTS

After 5 days treatment with 0.1% atRA, significant hypertrophy of the epidermis and dermis, neovascularization, and inflammatory cell invasion were seen in the skin of the db/db mice, but these effects were seen only weakly in db/+ mice. Wounds in atRA-treated db/db mice closed more rapidly than those in vehicle-treated db/db mice. KGF mRNA expression, which is usually significantly lower in db/db mice than in normal mice, in wounds of atRA-treated db/db mice on day 1 of treatment was as strong as in db/+ mice.

CONCLUSIONS

Pretreatment with atRA reversed the impaired wound healing in db/db mice.

Human skin organ cultures were established from <em>2</em>-mm punch biopsies and incubated under serum-free conditions in basal medium containing either 0.15 or 1.4 mM extracellular Ca<em>2</em>+. Organ cultures were treated with concentrations of sodium lauryl sulfate (SLS) that had previously been shown to support <em>growth</em> of human epidermal <em>keratinocytes</em> and human dermal fibroblasts in monolayer culture. Epidermal <em>growth</em> <em>factor</em> (EGF), alone and in combination with insulin and bovine pituitary extract, fetal bovine serum and all-trans retinoic acid (RA) were also examined for comparative purposes. The addition of SLS to culture medium containing low extracellular Ca<em>2</em>+ had no effect on the viability or histological appearance of the organ-cultured skin. Complete degeneration of the tissue occurred in the presence of SLS just as it did under control conditions. When SLS was added to culture medium containing high extracellular Ca<em>2</em>+, the basal layer of <em>keratinocytes</em> was much thinner than under control conditions. When EGF or EGF in combination with insulin and pituitary extract were utilized in place of SLS, identical results were obtained. That is, there was no preservation of the basal epithelial layer in the presence of low-Ca<em>2</em>+ culture medium and in the presence of high-Ca<em>2</em>+ culture medium, the basal layer was thinner than in control tissue. Virtually identical results were also obtained in medium containing 10% fetal bovine serum. In contrast, when RA was included in low-Ca<em>2</em>+ culture medium, the basal epithelium was maintained in a viable, histologically healthy condition. However, normal epithelial differentiation did not occur and the upper layers of the epidermis separated from the basal cells.(ABSTRACT TRUNCATED AT <em>2</em>50 WORDS)

The developing ovarian follicle is one of the most rapidly proliferating normal tissues in vivo. Mesenchymal-epithelial cell interactions between theca cells and granulosa cells are essential for this follicular expansion. Ovarian hormones (i.e. estrogen and LH) may promote follicular development by regulating the local production of mesenchymal inducer proteins that mediate theca cell-granulosa cell interactions. Recently, theca cells were shown to produce <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) that can act in a paracrine manner to stimulate granulosa cell <em>growth</em>. In this study, the developmental and hormonal regulation of KGF was examined during follicular development in the bovine ovary. Expression of KGF in theca cells and the KGF receptor (KGFR, or splice variant of the fibroblast <em>growth</em> <em>factor</em> family receptor family, FGFR-<em>2</em>) in granulosa cells was examined using RT-PCR. Both KGF and KGFR were detected throughout follicular development in small (<5 mm), medium (5-10 mm), and large (>10 mm) follicles. Quantitative RT-PCR assays were used to determine steady-state levels of KGF and KGFR messenger RNAs. Developmental regulation of KGF and KGFR was analyzed in freshly isolated theca cells and granulosa cells from small, medium, and large follicles. Observations demonstrated that expression of KGF (in theca cells) and KGFR (in granulosa cells) was highest in large follicles. These results suggest that KGF actions are important for the rapid proliferation of granulosa cells in large follicles. Estrogen and LH are the primary endocrine hormones that regulate theca cell function in vivo. Therefore, hormonal regulation of KGF was analyzed by treating serum-free theca cell cultures with estrogen and human CG (hCG, an LH agonist). Results showed that both estrogen and hCG stimulated KGF gene expression in theca cells. These results suggest that estrogen and LH may promote follicular <em>growth</em> (i.e. granulosa cell proliferation), in part, by stimulating the local production of KGF. Effects of KGF on granulosa cell differentiated functions were examined. Treatment with KGF reduced basal levels and FSH-stimulated levels of aromatase activity in bovine and rat granulosa cells. In addition, KGF inhibited the ability of hCG to stimulate progesterone production by granulosa cells. The inhibition of granulosa cell steroid production by KGF was likely the indirect effect of promoting cellular proliferation. Therefore, KGF directly stimulates granulosa cell proliferation and indirectly inhibits granulosa cell differentiated functions. Combined results suggest that theca cell production of KGF may be important for ovarian folliculogenesis. This is the first report of the regulation of KGF expression in the ovary. The developmental and hormonal regulation of KGF and KGFR during folliculogenesis provides evidence that KGF may be important for hormone-induced granulosa cell proliferation. As a result, KGF may be essential for establishing the microenvironment required for oocyte maturation in the ovary.

The transcription <em>factor</em> AP-<em>2</em>/promoter system is essential for gene expression associated with ectodermal development, particularly in the neural crest and skin. Three AP-<em>2</em> isoforms, alpha, beta, and gamma, exhibit a highly homologous structure, but their functions are considered to be different. Here, we report on the role of each AP-<em>2</em> isoform in complex <em>keratinocyte</em> biology including proliferation, differentiation, and carcinogenesis. The expression of AP-<em>2</em> was investigated immunohistochemically in serial skin sections from normal and psoriatic skin, and squamous cell carcinoma (SCC). AP-<em>2</em>alpha was present only in the nuclei of normal basal <em>keratinocytes</em>, but was significantly increased in lesional proliferating <em>keratinocytes</em> of both diseases. AP-<em>2</em>beta was completely absent from all skin samples except dermal sweat glands, whereas AP-<em>2</em>gamma was present homogeneously throughout the epidermis in normal and psoriatic skin as well as in the SCC lesion. Their restricted expression patterns correlated with in vitro DNA binding assays using selective <em>keratinocyte</em> gene promoters and three recombinant AP-<em>2</em> isoforms generated bacterially as glutathione S-transferase fusion protein. Epidermal <em>growth</em> <em>factor</em> receptor and basal keratin K14 promoters bound to AP-<em>2</em>alpha and AP-<em>2</em>gamma with similar affinities, whereas suprabasal keratin K1, type I transglutaminase, and involucrin promoters predominantly bound to AP-<em>2</em>gamma rather than AP-<em>2</em>alpha. In contrast, AP-<em>2</em>beta did not bind to any of the five promoters despite specific binding to the AP-<em>2</em> consensus probe. These results suggest that AP-<em>2</em>alpha is closely associated with <em>keratinocyte</em> proliferation and/or carcinogenesis rather than differentiation, while AP-<em>2</em>gamma is ubiquitous in all stages of <em>keratinocyte</em> biology. Taken together, three AP-<em>2</em> isoforms perform unique roles in the spatial and temporal expression of human <em>keratinocyte</em>-related genes, thereby maintaining epidermal homeostasis. Disruption of the epidermal AP-<em>2</em> balance may contribute to hyperproliferative conditions, such as psoriasis and SCC.

Because the level of extracellular Ca<em>2</em>+ is an important stimulus for differentiation of epidermal cells in vitro, we characterized the extracellular Ca(<em>2</em>+)-dependent transmembrane Ca<em>2</em>+ fluxes in BALB/MK mouse <em>keratinocytes</em>. Increasing levels of extracellular Ca<em>2</em>+, ranging from 0.07 to 1.87 mM, stimulated the rate of 45Ca<em>2</em>+ uptake into these cells 10- to 70-fold and doubled the rate of 45Ca<em>2</em>+ efflux. The divalent cations, Ni<em>2</em>+ and Co<em>2</em>+, were able to block the influx of Ca<em>2</em>+, but dihydropyridines and verapamil were not. Furthermore, 10 to 100 microM of the trivalent cation La3+ induced a dose-dependent <em>2</em>- to 100-fold increase of Ca<em>2</em>+ uptake, independently of the level of extracellular Ca<em>2</em>+. These observations suggest that <em>keratinocytes</em> possess a cell-surface "Ca(<em>2</em>+)-receptor," activation of which stimulates the influx of 45Ca<em>2</em>+ through a type of voltage-independent, receptor-operated Ca<em>2</em>+ channels. Epidermal <em>growth</em> <em>factor</em> induced an accumulation of 45Ca<em>2</em>+ of a much smaller magnitude than elevations of the level of extracellular Ca<em>2</em>+, without a detectable increase of Ca<em>2</em>+ efflux. Thus, the divergent cellular responses of <em>keratinocytes</em> to EGF and extracellular Ca<em>2</em>+ may be due, in part, to the distinct changes in transmembrane Ca<em>2</em>+ fluxes that these two stimuli generate. Treatment of cells with type beta transforming <em>growth</em> <em>factor</em> led to a gradual 6-fold increase of the Ca(<em>2</em>+)-activated rate of Ca<em>2</em>+ uptake over a period of 4 hours, but reduced the Ca<em>2</em>+ efflux by approximately 50% within 10 minutes. Thus, type beta transforming <em>growth</em> <em>factor</em> apparently stimulates Ca<em>2</em>+ influx indirectly, but may control the differentiation of <em>keratinocytes</em> by direct inhibition of Ca<em>2</em>+ efflux pumps.

The synthetic immunomodulator AS101[ammonium trichloro(dioxoethylene-o,o')tellurate] was previously found to protect cancer patients from chemotherapy-induced bone marrow toxicity and alopecia. Here we show that AS101 induces hair <em>growth</em> in nude and normal mice. AS101 possesses the dual ability to both induce anagen and retard spontaneous catagen in the C57BL/6 mouse model. Anagen induced by AS101 is mediated by <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF), as it is abrogated both in nude mice co-treated with AS101 plus neutralizing anti KGF antibodies and in AS101-treated transgenic mice expressing a dominant-negative KGF receptor transgene in basal <em>keratinocytes</em>. AS101 up-regulates KGF expression by activating the ras signaling pathway in cultured fibroblasts. AS101-induced delayed catagen is associated with inhibition of terminal differentiation marker expression both in nude and C57BL/6 mice epidermal follicular <em>keratinocytes</em> and in cultures of primary mouse follicular <em>keratinocytes</em> induced to differentiate. This activity is associated with relatively sustained elevation of p<em>2</em>1waf. Delayed expression of terminal differentiation markers was not induced by AS101 in follicular <em>keratinocytes</em> from p<em>2</em>1waf knockout mice. Because similar results were obtained with cultures of primary human <em>keratinocytes</em> and fibroblasts, preliminary case report studies revealed substantial hair <em>growth</em> when AS101 was topically applied on three adolescents who had remained alopeciac 1-<em>2</em> years after chemotherapy. The results emphasize the unique mode of action of AS101 and highlight its potential clinical use for treating certain types of alopecia.

BACKGROUND

Outer root sheath melanocytes (ORSM) are not yet routinely cultured and their biology is not known in detail because of their relatively low numbers in the hair follicle and their limited proliferative capacity in in vitro culture in routine media.

OBJECTIVE

To develop a method for culturing ORSM more easily and to investigate the length of telomeres and antigenic characteristics of ORSM compared with epidermal melanocytes (EM).

METHODS

Hair follicles were obtained from three Korean individuals during hair transplantation surgery. Single-cell suspensions of the outer root sheath were made and cultured in melanocyte <em>growth</em> medium with stem cell <em>factor</em>. After <em>2</em>1 days, second-passage outer root sheath <em>keratinocytes</em> (ORSK) (<em>2</em> x 10(4) mL(-1) MGM) were added into the culture plates. We studied the proliferation pattern, morphological and antigenic characteristics of ORSM for each passage of cultured cells, and observed ORSM telomere length.

RESULTS

We established an ORSM culture method using ORSK. Two morphologically different ORSM types were obtained in the primary cultures. At the end of primary culture, ORSM appeared as whitish-cream pellets. The proliferation pattern of ORSM showed a sigmoidal shape, the accumulated numbers of population doublings showed a plateau after approximately 5 months, and senescence occurred at approximately 33 +/- 5 accumulated population doublings. The length of ORSM telomeres continued to shorten as the cells proliferated. In contrast, EM showed a marked proliferation from the early proliferation period which formed a plateau pattern towards the later period, and the number of accumulated population doublings was estimated to be 18 +/- 5 after <em>2</em> months. ORSM in the primary culture reacted variably with l-dihydroxyphenylalanine (DOPA): some cells were DOPA negative, some DOPA positive. There were some different antigenic expressions of microphthalmia-associated transcription <em>factor</em> (MITF) showing cytoplasmic expression in ORSM and nuclear expression in EM. By nuclear extraction and Western blotting, we showed that MITF expression of ORSM was marked in the cytoplasm and minimal in the nucleus. Antigenic expression of MITF and Bcl-<em>2</em> gradually decreased with increasing passage number, whereas tyrosinase-related protein-1 expression did not change.

CONCLUSIONS

Culture of ORSM requires ORSK or ORSK-related factors; ORSM have greater proliferation potential and show different MITF antigenic expression compared with EM; and the length of ORSM telomeres shortens with repeated proliferation.

BACKGROUND

Ultraviolet (UV) irradiation to the skin causes apoptosis of <em>keratinocytes</em>. Melanocytes are more resistant to UV-induced apoptosis, due, in part, to high levels of antiapoptotic proteins such as Bcl-<em>2</em>. In vitro studies have shown that nerve <em>growth</em> <em>factor</em> (NGF), a neurotrophic polypeptide, is produced by <em>keratinocytes</em> and exerts a protective role for melanocytes by upregulating Bcl-<em>2</em>. The purpose of this study was to determine NGF and Bcl-<em>2</em> modulations in UV-irradiated human skin.

METHODS

Nine volunteers were irradiated with two minimal erythema doses using solar-simulated UV irradiation. Seventy-two hours post irradiation, skin biopsies were obtained from irradiated and sun-protected skin. The skin specimens were stained with anti-tyrosinase-related protein-1 monoclonal antibody IgG<em>2</em>a (Mel-5), anti-Bcl-<em>2</em> (monoclonal antibody IgG-kappa), and with anti-NGF (polyclonal antibody IgG).

RESULTS

NGF staining was identified within the cytoplasm of epidermal melanocytes, similar to the staining observed for TRP-1 and Bcl-<em>2</em>. While no significant difference in the number of TRP-1- and Bcl-<em>2</em>-positive melanocytes was observed between irradiated and non-irradiated skin within 7<em>2</em> h, the number of NGF-positive melanocytes decreased significantly, 7<em>2</em> h after UV irradiation (p < 0.0<em>2</em>4). NGF was also identified within <em>keratinocytes</em>, and while non-irradiated skin exhibited cytoplasmic NGF staining throughout the epidermis, NGF staining was reduced in the lower epidermal layers after UV irradiation.

CONCLUSIONS

This is the first in vivo study showing NGF to be present in melanocytes, as well as showing modulations of NGF and Bcl-<em>2</em> in melanocytes, following solar-simulated UV irradiation.

(S)-1-[3-Hydroxy-<em>2</em>-(phosphonylmethoxy)propyl]cytosine (HPMPC) is a nucleoside phosphonate analog which in its active diphosphorylated form is known to inhibit herpesvirus DNA polymerase. In this study, we have demonstrated that, in a dose-dependent manner, this compound irreversibly suppressed proliferation of cells infected with human papillomavirus (HPV), which does not possess a viral DNA polymerase. To elucidate the mechanism of cell <em>growth</em> inhibition, cell cycle indicator-regulator expression, thymidine incorporation, transcript levels of apoptosis <em>factors</em>, and anabolic products of HPMPC following drug treatment were evaluated. HPMPC treatment reduced WAF1 (p<em>2</em>1) levels independent of those of p53, while proliferating cell nuclear antigen increased. However, in comparison to controls, HPMPC-treated cells displayed a decrease in thymidine incorporation, indicating an inhibition of host DNA polymerase activity. In normal primary <em>keratinocytes</em>, HPMPC predominantly accumulated in the form of the choline adduct HPMPCp-choline. However, in HPV type 16-transformed <em>keratinocytes</em>, HPMPCpp was the most abundant anabolic product, with little HPMPCp-choline having formed. The data imply that an unrecognized viral <em>factor</em> is modulating the conversion of nucleotides, including HPMPC, to the triphosphorylated form.

S100A11, a member of the family of S100 proteins, is a dimmer, each monomer of which has two EF-hands. Expression of S100A11 is ubiquitous in various tissues at different levels, with a high expression level in the skin. We have analyzed functions of S100A11 mainly in normal human <em>keratinocytes</em> (NHK) as a model cell system of human epithelial cells. High Ca(<em>2</em>+) and transforming <em>growth</em> <em>factor</em>-β (TGF-β), two representative <em>growth</em> suppressors for NHK, need a common S100A11-mediated pathway in addition to unique pathways (NFAT1-mediated pathway for high Ca(<em>2</em>+) and Smad-mediated pathway for TGF-β) for exhibiting a <em>growth</em> inhibitory effect. S100A11 has another action point for <em>growth</em> suppression in NHK. Annexin A1 (ANXA1) complexed with S100A11 efficiently binds to and inhibits cytosolic phospholipase A<em>2</em> (cPLA<em>2</em>), the activity of which is needed for the <em>growth</em> of NHK. On exposure of NHK to epidermal <em>growth</em> <em>factor</em> (EGF), ANXA1 is cleaved at 1<em>2</em>Trp, and this truncated ANXA1 loses binding capacity to S100A11, resulting in maintenance of an active state of cPLA<em>2</em>. On the other hand, we found that S100A11 is actively secreted by NHK. Extracellular S100A11 acts on NHK to enhance the production of EGF family proteins, resulting in <em>growth</em> stimulation. These findings indicate that S100A11 plays a dual role in <em>growth</em> regulation, being suppressive in cells and being promotive from outside of cells.