Cutaneous squamous cell carcinoma (cSCC) is one of the most common metastatic skin cancers with increasing incidence. We examined the roles of complement component C3 and complement <em>factor</em> B (CFB) in the <em>growth</em> of cSCC. Analysis of cSCC cell lines (n = 8) and normal human epidermal <em>keratinocytes</em> (n = 11) with real-time quantitative PCR and Western blotting revealed up-regulation of C3 and CFB expression in cSCC cells. Immunohistochemical staining revealed stronger tumor cell-specific labeling for C3 and CFB in invasive cSCCs (n = 71) and recessive dystrophic epidermolysis bullosa-associated cSCCs (n = 11) than in cSCC in situ (n = 69), actinic keratoses (n = 63), and normal skin (n = 5). Significant up-regulation of C3 and CFB mRNA expression was noted in chemically induced mouse cSCCs, compared to benign papillomas. Knockdown of C3 and CFB expression inhibited migration and proliferation of cSCC cells and resulted in potent inhibition of extracellular signal-regulated kinase 1/<em>2</em> activation. Knockdown of C3 and CFB markedly inhibited <em>growth</em> of human cSCC xenograft tumors in vivo. These results provide evidence for the roles of C3 and CFB in the development of cSCC and identify them as biomarkers and potential therapeutic targets in this metastatic skin cancer.

OBJECTIVE

To evaluate mechanisms underlying modulation of inflammatory chemokines in primary human keratinocytes (normal human epidermal keratinocytes) and repair-related processes in wound models by plant polyphenols (PPs) with antioxidant and superoxide scavenging properties (verbascoside [Vb], resveratrol [Rv], polydatin [Pd], quercetin [Qr], and rutin).

RESULTS

Epidermal growth factor receptor (EGFR)-controlled chemokines CXCL8/interleukin 8 (IL-8), CCL2/monocyte chemotactic protein-1 (MCP-1), and CXCL10/interferon gamma-produced protein of 10 kDa (IP-10) were modulated by transforming growth factor alpha (TGF-α) and by the tumor necrosis factor alpha/interferon gamma combination (T/I). EGFR phosphorylation, nuclear translocation, and downstream cytoplasmic signaling pathways (extracellular regulation kinase [ERK]1/2, p38, STAT3, and PI-3K) were studied. All PPs did not affect TGF-α-induced STAT3 phosphorylation, whereas they suppressed T/I-activated NFkappaB and constitutive and T/I-induced but not TGF-α-induced ERK1/2 phosphorylation. Vb and Qr suppressed total EGFR phosphorylation, but they synergized with TGF-α to enhance nuclear accumulation of phosphorylated EGFR. Vb strongly inhibited TGF-α-induced p38 phosphorylation and T/I-induced NFkappaB and activator protein-1 (AP-1) binding to DNA. Vb was an effective inhibitor of T/I-stimulated chemokine synthesis, and it accelerated scratch wound healing in vitro. Anti-inflammatory and wound healing activities of Vb were confirmed in vivo in the full-thickness excision wound. Although Pd and Rv did not affect EGFR activation/translocation, they and Qr synergized with TGF-α and T/I in the induction of IL-8 transcription/synthesis while opposing enhanced MCP-1 and IP-10 transcription/synthesis connected with pharmacologically impaired EGFR functioning.

METHODS

PPs perturb the EGFR system in human keratinocytes, and this effect may be implicated in the regulation of inflammatory and repair-related processes in the skin.

CONCLUSIONS

Anti-inflammatory and wound healing effects of PPs depend on their interaction with EGFR-controlled cytoplasmic and nuclear pathways rather than on their direct redox properties.

BACKGROUND

Nanoparticles are widely used in different technological fields, one of which is medicine. Because of their antibacterial properties, silver nanoparticles (AgNPs) are used in several types of wound dressings for the treatment of burns and nonhealing wounds, but their influence on each component of the wound-healing process remains unclear. In the present study, we evaluated the effects of AgNPs on normal human dermal fibroblasts (NHDFs) and normal human epidermal keratinocytes (NHEKs). Both cell types are important for wound healing, including with regard to inflammation, proliferation and tissue remodeling. Each phase of wound healing can be characterized by the secretion of cytokines, chemokines and growth factors.

METHODS

The production of inflammatory parameters (tumor necrosis factor α [TNF-α], interleukin-6 [IL-6], IL-8 and IL-12 and cyclooxygenase-2 [COX-2]), angiogenesis parameters (vascular endothelial growth factor [VEGF], granulocyte macrophage colony-stimulating factor) and matrix metalloproteinases (MMP-1, MMP-2, MMP-3 and MMP-9) by NHDFs and NHEKs were examined by ELISA or Western blot after 24 and 48 hours of incubation with AgNPs.

RESULTS

We found that AgNPs decreased some inflammatory cytokines (TNF-α and IL-12) and growth factors (VEGF) that were produced by NHDFs and NHEKs after 24 and 48 hours and decreased the expression of COX-2 after 24 hours but only at the highest concentration of AgNPs (25 parts per million).

CONCLUSIONS

The results indicate that NHEKs are more susceptible to the application of AgNPs than NHDFs, and AgNPs may be useful for medical applications for the treatment of wounds.

Exposure to ultraviolet (UV) irradiation has detrimental effects on skin accompanied by the increased metabolism of hyaluronan (HA), a linear polysaccharide important for the normal physiological functions of skin. In this study, the modulation of human <em>keratinocyte</em> response to UVB irradiation by HA (970 kDa) was investigated. Immortalized human <em>keratinocytes</em> (HaCaT) were irradiated by a single dose of UVB and immediately treated with HA for 6 and <em>2</em>4 h. The irradiation induced a significant decrease in the gene expression of CD44 and toll-like receptor <em>2</em> 6 h after irradiation. The expressions of other HA receptors, including toll-like receptor 4 and the receptor for HA-mediated motility, were not detected in either the control or UVB-irradiated or HA-treated HaCaT cells. UVB irradiation induced a significant decrease in the gene expression of HA synthase-<em>2</em> and hyaluronidase-<em>2</em> 6 h after irradiation. The expressions of HA synthase-3 and hyaluronidase-3 were not significantly modulated by UV irradiation. Interestingly, HA treatment did not significantly modulate any of these effects. In contrast, HA significantly suppressed UVB-induced pro-inflammatory cytokine release including interleukin-6 and interleukin-8. Similarly, HA treatment reduced the UVB-mediated production of transforming <em>growth</em> <em>factor</em> β1. HA treatment also significantly reduced the UV irradiation-mediated release of soluble CD44 into the media. Finally, HA partially, but significantly, suppressed the UVB-induced decrease in cell viability. Data indicate that HA had significant protective effects for HaCaT cells against UVB irradiation.

Wound healing is a complex process of which <em>growth</em> and motility are essential features. The aim of this study was to search for <em>keratinocyte</em>-derived secreted <em>factors</em> that may play a role in these mechanisms, and their corresponding receptors. <em>Growth</em> and motility <em>factors</em> were purified from conditioned medium from cultured primary <em>keratinocytes</em>. Receptor and <em>growth</em> <em>factor</em> expression profiles were investigated by immunohistochemical, western blotting, and in situ hybridization analysis on cultured <em>keratinocytes</em> and tissue sections derived from chronic wounds. The most potent autocrine <em>growth</em> <em>factor</em> for <em>keratinocytes</em>, which it was possible to purify and sequence from <em>keratinocyte</em>-conditioned medium, is amphiregulin. Its receptor HER-1 is up-regulated on the membranes of <em>keratinocytes</em> lining the edge of the wound. From the same <em>keratinocyte</em>-conditioned medium, heregulin-alpha was purified as a potent motility <em>factor</em> for <em>keratinocytes</em>. Its receptor is HER-3, which is up-regulated on the membranes of <em>keratinocytes</em> lining the edge of the wound and on <em>keratinocytes</em> that had migrated towards the centre of the wound. HER-4 - another receptor for heregulin-alpha - is weakly present in occasional cells near the edge of the wound. The co-receptor for HER-3 and HER-4 is HER-<em>2</em>/neu, which is also present in epidermal cells but not overexpressed. This study shows that heregulin-alpha is a potent motility <em>factor</em> for normal epithelial cells and that it plays a central role in the process of wound healing of stratified epithelia. Heregulin-alpha has already been shown to be the motility <em>factor</em> leading to migration of HER-<em>2</em>/neu-overexpressing breast cancer cells. The role of amphiregulin as a <em>growth</em> <em>factor</em> and of heregulin-alpha as a motility <em>factor</em> for <em>keratinocytes</em> in epidermal and mucosal wound healing parallels their motility and <em>growth</em> induction in carcinogenesis.

<em>Keratinocytes</em> contribute to melanocyte transformation by affecting their microenvironment, in part through the secretion of paracrine <em>factors</em>. Here we report a loss of expression of nuclear receptor RXRα in epidermal <em>keratinocytes</em> 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 <em>keratinocyte</em>-derived mitogenic <em>growth</em> <em>factors</em> (Et-1, Hgf, Scf, α-MSH and Fgf <em>2</em> ) 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 <em>keratinocytes</em>. These studies demonstrate the contribution of keratinocytic paracrine signaling during the cellular transformation and malignant conversion of melanocytes.

Hair bulb <em>keratinocytes</em> generate one of the few "immune privileged" tissue compartments of the mammalian organism by suppressing classical MHC class I (MHC Ia) antigens. Expression of non-classical MHC class I (MHC Ib) antigens in the follicle has been found, but only in its distal epithelium. Here, we have defined when during murine hair follicle morphogenesis these peculiar MHC Ia and Ib expression patterns are established, how they change during the murine hair cycle, and how different MHC I modulatory agents alter follicular MHC Ia and Ib expression in vivo. During neonatal hair follicle morphogenesis in C57BL/6 mice, distal follicle <em>keratinocytes</em> began to express MHC Ia (H<em>2</em>b) only late in development. The MHC Ib antigens, Qa-1 and Qa-<em>2</em>, did not become visible until the initiation of follicle cycling, with Qa-1 expression being more widespread than that of Qa-<em>2</em>. H<em>2</em>b, Qa-1, and TAP-1 immunoreactivity on previously negative <em>keratinocytes</em> of the proximal anagen hair bulb was upregulated by intradermal injection of the proinflammatory cytokine interferon-gamma, but not by tumor necrosis <em>factor</em>-alpha or interleukin-1beta. Injection of the reportedly MHC class I downregulating agents interleukin-10, insulin-like <em>growth</em> <em>factor</em>-1, transforming <em>growth</em> <em>factor</em>-beta, alpha-melanocyte stimulating hormone, or dexamethasone, however, all failed to downregulate constitutive or interferon-gamma-induced follicular MHC Ia expression. This shows that the hair follicle is a previously unrecognized site of Qa-1 expression and that interferon-gamma is a key regulator of follicular MHC I expression in vivo. It also suggests that the developmental and immunologic controls of MHC I expression by follicle <em>keratinocytes</em> differ from those of other epithelial cells.

Insulin-like <em>growth</em> <em>factor</em>-I (IGF-I) activation of phosphoinositol 3-kinase (PI3K) is an essential pathway for <em>keratinocyte</em> migration that is required for epidermis wound healing. We have previously reported that activation of Galpha((q/11))-coupled-P<em>2</em>Y(<em>2</em>) purinergic receptors by extracellular nucleotides delays <em>keratinocyte</em> wound closure. Here, we report that activation of P<em>2</em>Y(<em>2</em>) receptors by extracellular UTP inhibits the IGF-I-induced p110alpha-PI3K activation. Using siRNA and pharmacological inhibitors, we demonstrate that the UTP antagonistic effects on PI3K pathway are mediated by Galpha((q/11))-and not G((i/o))-independently of phospholipase Cbeta. Purinergic signaling does not affect the formation of the IGF-I receptor/insulin receptor substrate-I/p85 complex, but blocks the activity of a membrane-targeted active p110alpha mutant, indicating that UTP acts downstream of PI3K membrane recruitment. UTP was also found to efficiently attenuate, within few minutes, the IGF-I-induced PI3K-controlled translocation of the actin-nucleating protein cortactin to the plasma membrane. This supports the UTP ability to alter later migratory events. Indeed, UTP inhibits <em>keratinocyte</em> spreading and migration promoted by either IGF-I or a membrane-targeted active p110alpha mutant, in a Galpha(q/11)-dependent manner both. These findings provide new insight into the signaling cross-talk between receptor tyrosine kinase and Galpha((q/11))-coupled receptors, which mediate opposite effects on p110alpha-PI3K activity and <em>keratinocyte</em> migration.

The pathogenic mechanism underlying the hyperproliferation of <em>keratinocytes</em> in psoriasis is still not completely clarified. The production of cytokines released by activated T lymphocytes infiltrating the upper dermis probably has a crucial role. Even dermal fibroblasts can participate in the process through the secretion of <em>growth</em> <em>factors</em>, and some studies have reported an increased expression of the insulin-like <em>growth</em> <em>factor</em> 1. Few studies, however, have focused on the possible involvement of the <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF/FGF-7) and the fibroblast <em>growth</em> <em>factor</em> 10 (FGF-10/KGF-<em>2</em>), which are secreted by fibroblasts and stimulate <em>keratinocyte</em> proliferation acting through a receptor specifically expressed by epithelial cells. The aim of this study was to investigate the expression of KGF and FGF-10 on the skin of patients with psoriasis by immunohistochemical analysis and to evaluate the correlation with the lymphocyte infiltrate and the epidermal proliferation. Immunostaining for KGF and FGF-10 showed that both the <em>growth</em> <em>factors</em> are upregulated in the upper dermis of psoriatic skin, and that the expression is correlated with the presence of T-cell infiltrate and with <em>keratinocyte</em> proliferation. Our data suggest that in psoriatic lesions activated lymphocytes can stimulate fibroblasts to produce KGF and FGF-10, which in turn contribute to sustain the hyperproliferative status of the <em>keratinocytes</em>.

The CXC-chemokines Groalpha and interleukin-8 (IL-8) are well characterized <em>growth</em> <em>factors</em> for melanoma cells. Here the constitutive expression of Groalpha, IL-8 and their receptors (CXCR1 and CXCR<em>2</em>) as well as their functional involvement in the proliferation response were analyzed in normal <em>keratinocytes</em> and epidermoid carcinoma cell lines A431 and KB. Flow cytometric measurements, ELISA and semi-quantitative RT-PCR revealed low constitutive protein secretion and mRNA expression of both CXC-chemokines as well as CXCR1 and <em>2</em> in normal <em>keratinocytes</em>, whereas significant higher levels of CXC-chemokines and CXCR<em>2</em> were deteced in epidermoid carcinoma cells. Proliferation of epidermoid carcinoma cells could be induced by CXC-chemokines and constitutive proliferation could be inhibited by neutralizing antibodies against CXC-chemokines and CXCR<em>2</em>. These studies indicate that constitutive Groalpha, IL-8 and CXCR<em>2</em> protein expression enable an autocrine <em>growth</em> mechanism in epidermoid carcinoma cells.

OBJECTIVE

The aim of the present study was to quantify the protective efficacy of recombinant human keratinocyte growth factor (rHuKGF) in oral mucosa.

METHODS

Mouse tongue mucosal ulceration was analyzed as the clinically relevant end point. Fractionated irradiation of the snout with 5 daily fractions of 3 Gy was followed by graded test doses, given to a test area of the lower tongue, on Day 7. rHuKGF was injected s.c. in daily doses of 5 mg/kg before radiotherapy, during radiotherapy, over the weekend break, or a combination. Moreover, single rHuKGF injections (5 or 15 mg/kg) were given on Day -1 or on Day 4.

RESULTS

In a single-dose control experiment, the ED50, i.e., the dose after which ulcer induction is expected in 50% of the mice, was 10.9 +/- 0.7 Gy. Fractionated irradiation without keratinocyte growth factor rendered an ED50 for test irradiation of 5.6 +/- 3.7 Gy. Keratinocyte growth factor increased the ED50 values to 7.8 +/- 3.3 Gy (Days -3 to -1, p = 0.01), 8.3 +/- 1.6 Gy (Days -4 to -2, p = 0.0008), 10.5 +/- 1.4 Gy (Days 0 to +2, p = 0.0002), 11.0 +/- 0.5 Gy (Days 0 to +4, p = 0.002), 10.6 +/- 1.4 Gy (Days +4 to +6, p = 0.0021), 10 +/- 0.07 (Days -3 to +1, p = 0.0001) or 11.0 +/- 0.02 (Days +4 to +8, p = 0.0001). This is equivalent to compensation of approximately 1.5 fractions of 3 Gy when rHuKGF is given before radiotherapy and 3-4 fractions in all other protocols by rHuKGF treatment. Single rHuKGF injections were similarly (5 mg/kg) or more (15 mg/kg) effective.

CONCLUSIONS

In conclusion, these results indicate a marked increase in oral mucosal radiation tolerance by rHuKGF, which is most pronounced if the growth factor is applied during fractionated radiotherapy. The effect seems to be based on complex mechanisms, predominantly changes in both epithelial proliferation and differentiation processes.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em>-<em>2</em> (KGF-<em>2</em>), also described as fibroblast <em>growth</em> <em>factor</em>-10 (FGF-10), is a member of the fibroblast <em>growth</em> <em>factor</em> family. KGF-<em>2</em> shares 57 per cent sequence homology to previously reported KGF-1 (FGF-7). In skin, both <em>growth</em> <em>factors</em> are expressed in the dermal compartment. KGF-1 and KGF-<em>2</em> bind to the same receptor with high affinity, the KGFR isoform of FGFR<em>2</em>, which is exclusively expressed by epithelial cells. This study examines the in vivo function of topically applied KGF-<em>2</em> on wound healing using an ischaemia-impaired rabbit dermal ulcer model, in young and aged animals. Histological analysis of the wounds showed that KGF-<em>2</em> significantly promoted re-epithelialization in both young and old animals. Similar results have been observed with KGF-1 in this model. In addition, KGF-<em>2</em> enhanced granulation tissue formation in both young and old rabbits, a biological effect not found with KGF-1, suggesting a possible indirect mechanism which enhances neo-granulation tissue formation. Immunohistological staining of day 7 wounds with proliferating cell nuclear antigen (PCNA) antibody demonstrated a significant increase of dermal cell proliferation in KGF-<em>2</em>-treated wounds compared with placebo wounds. These results suggest a mesenchymal-epithelial interaction that is mediated by a paracrine feedback loop of KGF-<em>2</em>. Because of the wound healing impairment observed with ageing, the wound healing response to KGF-<em>2</em> was also studied in ischaemic wounds of aged animals. Administration of KGF-<em>2</em> led to significant stimulation of epithelial <em>growth</em> and granulation tissue formation. The effects seen in the old animals were delayed compared with the young animals. Lastly, the effect of KGF-<em>2</em> was examined in a rabbit model of scar formation. Quantification of scar elevation index showed no significant differences in scar formation when KGF-<em>2</em> was compared with buffer placebo. Compared with other <em>growth</em> <em>factors</em>, including KGF-1 and TGF-beta which have previously been examined in these models, KGF-<em>2</em> is the most effective and causes no obvious scarring.

Human epithelial cells are permanently stimulated by external mechanical forces. The present in vitro study suggests that <em>keratinocytes</em> respond to mechanical strain by a coordinated spatial and functional utilization of beta1-integrins and the epidermal <em>growth</em> <em>factor</em> receptor (EGFR) with impact to the adhesion properties. It was found that a single mechanical stretch applied to HaCaT <em>keratinocytes</em> elevates the substrate adhesion, in particular to fibronectin and collagen type IV but not to laminin indicating the relevance of beta1-integrins in this process. This was confirmed using a functional blocking antibody directed against beta1-integrins which reversed the stretch-induced adhesion. Furthermore, mechanical stretch gives rise to a rapid redistribution of beta1-integrins in clusters on the basal cell membrane, without changing the overall amount of this particular integrin subset. Concomitantly, the EGFR co-localizes with beta1-integrin suggesting a functional cooperation of both membrane proteins in mechano-signaling. This is corroborated by data showing that stretch-induced activation of the EGFR and the downstream element extracellular regulated kinase 1/<em>2</em> (ERK1/<em>2</em>) is reversed by preincubation with beta1-integrin antibodies. Vice versa, blocking the EGFR using a specific inhibitor abrogates stretch-induced ERK1/<em>2</em> activation. In summary, these results show a functional cooperation of beta1-integrins and EGFR in the adhesion complex supporting the transmission of stretch-induced signals.

Vitiligo is a puzzling disorder characterized by a disappearance of epidermal and/or follicular melanocytes by unknown mechanisms. This very common disorder involving 1-4% of the world population is thus of great importance for the practicing dermatologist. The cellular and molecular mechanisms leading to the destruction of melanocytes in this disorder have not yet been elucidated, making it of major interest for the cell biologist involved in melanocyte research. Recent advances in this field, due largely to the availability of techniques for culturing normal human melanocytes, opened new perspectives in the understanding of vitiligo. Although vitiligo has long been considered a disorder confined to the skin, there is now good evidence that it also involves the extracutaneous compartment of the "melanocyte organ." It is also clear that vitiligo is not only a melanocyte disorder, but that it also involves cells, such as <em>keratinocytes</em> and Langerhans cells, found in the epidermis and follicular epithelium. The three prevailing theories of the pathogenesis of vitiligo are the immune hypothesis, the neural hypothesis, and the self-destruct hypothesis. New hypotheses suggest that vitiligo may be due to (1) a deficiency in an unidentified melanocyte <em>growth</em> <em>factor</em>, (<em>2</em>) an intrinsic defect of the structure and function of the rough endoplasmic reticulum in vitiligo melanocytes, (3) abnormalities in a putative melatonin receptor on melanocytes and (4) a breakdown in free radical defense in the epidermis. None of these hypotheses has been demonstrated, and according to the available data, it is likely that the loss of epidermal and follicular melanocytes in vitiligo may be the result of several different pathogenetic mechanisms.

In this study, we determined regulation and function of the suppressor of cytokine signaling (SOCS)-3 in acute and impaired murine skin repair. Upon skin injury, SOCS-3 was induced and expressed during the inflammatory phase of repair. SOCS-3 protein expression was localized in a subset of non-proliferating <em>keratinocytes</em> within the developing wound margin epithelia. <em>Growth</em> <em>factors</em> (EGF, transforming <em>growth</em> <em>factor</em>-alpha), nitric oxide (NO), and pro-inflammatory cytokines were inducers of SOCS-3 mRNA and protein expression in cultured human (HaCaT) and primary murine <em>keratinocytes</em>. Stable overexpression of SOCS-3 in HaCaT <em>keratinocytes</em> interfered with cytokine-induced signal transducers and activators of transcription-3 phosphorylation and inhibited serum-stimulated proliferation of the cells. Moreover, overexpression of SOCS-3 led to final differentiation of <em>keratinocytes</em>, which was comparable to the Ca(<em>2</em>+)-induced differentiation process in the cells. Finally, we determined SOCS-3 expression in two models of impaired skin repair: NO-deficient and diabetic wound healing. In line with observations from normal repair and SOCS-3 overexpression experiments, reduced <em>keratinocyte</em> proliferation within atrophied neo-epithelia in both models of impaired healing was associated with a marked increase in SOCS-3-expressing wound <em>keratinocytes</em>. In summary, this study suggests a potential novel function of SOCS-3 in regulating <em>keratinocyte</em> proliferation and differentiation in vitro and during skin repair in vivo.

Heat shock proteins (hsp) have important roles in the regulation and protection of both prokaryotic and eukaryotic cells, especially during environmental stress. Hsps are also important bacterial virulence <em>factors</em>. We investigated whether bacterial hsp60 can alter epithelial cell mitogen-activated protein kinase (MAPK) signaling and cell proliferation. Human skin <em>keratinocytes</em> (HaCaT cell line) were cultured in the presence of hsp60 purified from Actinobacillus actinomycetemcomitans, an important oral pathogen. Protein kinases in the ERK1/<em>2</em> and p38 MAPK signaling pathways were probed with kinase-specific and phosphorylation-site-specific antibodies on Western blots. In quiescent cultures, hsp60 increased ERK1/<em>2</em> phosphorylation in a sustained manner and p38 phosphorylation transiently. Hsp60 also increased epithelial cell proliferation by about 30%. Inhibition of the ERK1/<em>2</em> pathway by PD 98059 (a MEK1 inhibitor) reversed partially ERK1/<em>2</em> phosphorylation and totally cell proliferation indicating that the ERK1/<em>2</em> MAPK pathway is involved in the hsp60-induced cell <em>growth</em>. This was supported by findings that hsp60 stimulated phosphorylation of RSK1/<em>2</em> and cyclic AMP response element-binding protein and increased expression of transcription <em>factors</em> c-Jun and c-Fos. Recombinant human hsp60 did not alter ERK1/<em>2</em> or p38 phosphorylation and had no effect on epithelial cell proliferation. Inhibition of p38 MAPK pathway by SB <em>2</em>03580 increased both ERK1/<em>2</em> phosphorylation and cell proliferation demonstrating that the inhibitor can either directly or indirectly activate the ERK1/<em>2</em> MAPK pathway. The results show that exogenous bacterial hsp60 is able to activate ERK1/<em>2</em> phosphorylation and thereby cause increased epithelial proliferation. In case of mucosal infection this effect may either lead to increased wound repair or participate in the pathological mechanism of some bacterial diseases that involve increased epithelial proliferation.

OBJECTIVE

To evaluate the safety of repifermin (<em>keratinocyte</em> <em>growth</em> <em>factor</em>-<em>2</em>) administered before and after autologous hematopoietic stem cell transplantation (auto-HSCT). A preliminary analysis of the ability of <em>keratinocyte</em> <em>growth</em> <em>factor</em>-<em>2</em> to prevent mucositis was also done.

METHODS

Forty-two patients received intravenous repifermin (<em>2</em>5 microg/kg or 50 microg/kg) or placebo for 3 days before their auto-HSCT conditioning regimen and for up to 10 days after auto-HSCT. Within each dose level, 14 patients were randomized to repifermin and 7 patients to placebo. Clinical evaluations of mucositis were scheduled before auto-HSCT conditioning regimen, on the day of transplant, and three times per week until mucositis resolved.

RESULTS

In general, the incidence of adverse events was similar for patients treated with repifermin and placebo. No clinically meaningful differences were noted among treatment groups for clinical laboratory variables. Treatment groups experienced similar time to engraftment. The frequency of Grade <em>2</em> to 4 mucositis was 100% for patients in the placebo group, 64% for patients in the <em>2</em>5 microg/kg group (P = 0.041 versus placebo), and 50% for patients in the 50 microg/kg group (P = 0.006 versus placebo). Results of other endpoints, including pain on swallowing and use of pain medication specifically for mucositis, suggested a better outcome for patients in the 50 microg/kg group compared with the placebo and <em>2</em>5 microg/kg groups.

CONCLUSIONS

Repifermin was well tolerated. Repifermin given before and after auto-HSCT seems to be active in reducing mucositis, but a larger trial will be necessary to determine the efficacy of repifermin with this dose schedule.

OBJECTIVE

Several physical agents such as low-energy lasers have been used in the treatment of chronic skin ulcers. This study was performed to investigate potential effects of a newly-developed, specific near-infrared light source on wound repair.

METHODS

Cultured human <em>keratinocytes</em>, endothelial cells and fibroblasts were exposed to the light, and the production of transforming <em>growth</em> <em>factor</em> (TGF)-beta1 and matrix metalloproteinase (MMP)-<em>2</em> was examined by enzyme immunoassay, zymography and reverse transcription polymerase chain reaction. Incisional wounds were created in ICR and db/db diabetic mice and the effect of irradiation on wound closure was followed photographically.

RESULTS

The TGF-beta1 and MMP-<em>2</em> content of the medium of cultured cells was significantly elevated after irradiation. The amount of MMP-<em>2</em> mRNA extracted from irradiated fibroblasts was also upregulated. Negative results in thermal controls suggested that the action of the light was athermic in nature. In animal models, the rate of wound closure was significantly accelerated by repeated exposures.

CONCLUSIONS

Near-infrared irradiation potentially enhances the wound healing process, presumably by its biostimulatory effects.

Collagen VII, the major component of cutaneous anchoring fibrils is expressed at a low level by normal human <em>keratinocytes</em> and fibroblasts in vitro. In cocultures of these two cell types, signals from fibroblasts enhance expression of collagen VII by <em>keratinocytes</em> and vice versa. In this study, the effects of a possible mediator of such a stimulation, transforming <em>growth</em> <em>factor</em>-beta (TGF-beta), were investigated. Its effect on the expression and deposition of the highly insoluble collagen VII was assessed in a semiquantitative manner by a newly developed enzyme-linked immunoassay which is based on immunoblotting. In <em>keratinocyte</em> monocultures, 0.5-<em>2</em>0 ng/ml of TGF-beta <em>2</em> induced a dose-dependent stimulation of collagen VII expression as measured per microgram of DNA. The maximal enhancement was about sevenfold compared to controls. The effect of TGF-beta <em>2</em> was observed already after 1<em>2</em> h, with a steady increase at least up to 3 d. As previous studies have implicated, untreated cocultures of <em>keratinocytes</em> and fibroblasts exhibited a higher basic level of collagen VII expression, which could be further stimulated about twofold by TGF-beta <em>2</em>. Fibroblasts alone synthesized very minor quantities of collagen VII and could be only weakly stimulated by TGF-beta <em>2</em>. This <em>growth</em> <em>factor</em> seems a specific enhancer of collagen VII since the expression of laminin, collagen IV, as well as total protein was increased to a much lesser extent. Our data suggest that TGF-beta may be an important mediator of epithelial-mesenchymal interactions and may regulate the synthesis of the anchoring fibrils at the skin basement membrane zone.

We generated a mouse strain lacking protein kinase Calpha (PKCalpha) and evaluated the significance of the enzyme in epithelial hyperplasia and tumor formation. PKCalpha-deficient mice exhibited increased susceptibility to tumor formation in two-stage skin carcinogenesis by single application of 7,1<em>2</em>-dimethylbenz(a)anthracene (DMBA) for tumor initiation and repeated applications of 1<em>2</em>-O-tetradecanoylphorbol-13-acetate (TPA) for tumor promotion. Tumor formation was not enhanced by DMBA or TPA treatment alone, suggesting that PKCalpha suppresses tumor promotion. However, the severity of epidermal hyperplasia induced by topical TPA treatment was markedly reduced. In mutant mice, the number of 5-bromo-<em>2</em>'-deoxyuridine-labeled epidermal basal <em>keratinocytes</em> increased 16 to <em>2</em>4 hours after topical TPA treatment as in the case of wild-type mice, but significantly decreased at 36 and 48 hours. Furthermore, the regenerating epithelium induced by skin wound significantly decreased in thickness but was not structurally impaired. The enhanced tumor formation may not be associated with epidermal hyperplasia. The induction levels of epidermal <em>growth</em> <em>factor</em> (EGF) receptor ligands, tumor <em>growth</em> <em>factor</em> alpha (TGF-alpha), and heparin-binding EGF-like <em>growth</em> <em>factor</em>, in the skin of mutant mice by TPA treatment were significantly lower than those in the skin of wild-type mice. PKCalpha may regulate the supply of these EGF receptor ligands in basal <em>keratinocytes</em>, resulting in a reduced epidermal hyperplasia severity in the mutant mice. We propose that PKCalpha positively regulates epidermal hyperplasia but negatively regulates tumor formation in two-stage skin carcinogenesis.

Phenolic compounds used in pharmaceutical and industrial products can cause irritant contact dermatitis. We studied the effects of resorcinol, phenol, 3,5-xylenol, chloroxylenol, and 4-hexyl-resorcinol on normal human epidermal <em>keratinocytes</em> and dermal fibroblasts for cytotoxicity and cytokine release, determined by 3-(4,5-dimethylthiazol-<em>2</em>-yl)-<em>2</em>,5-diphenyl tetrazolium bromide methodology and enzyme-linked immunosorbent assay, respectively. An inverse correlation between phenol concentrations causing a 50% reduction in <em>keratinocyte</em> and fibroblast viability at <em>2</em>4 h and their octanol water-partition coefficients (i.e., hydrophobicity) was observed. 3,5-xylenol, chloroxylenol, hexyl-resorcinol, and sodium dodecyl sulfate, but not resorcinol or phenol, induced release of interleukin-1alpha from <em>keratinocytes</em> at cytotoxic concentrations. Variable release of tumor necrosis <em>factor</em>-alpha and interleukin-8 from <em>keratinocytes</em> occurred only at toxic threshold concentrations of the phenols or sodium dodecyl sulfate. Subtoxic concentrations of phenols or sodium dodecyl sulfate did not induce cytokine release from <em>keratinocytes</em>. Neither the phenols nor sodium dodecyl sulfate induced release of the chemokines interleukin-8, <em>growth</em>-related oncogene-alpha or monocyte chemotactic protein-1 from fibroblasts. Conditioned media from <em>keratinocytes</em> treated with cytotoxic concentrations of 3,5-xylenol, chloroxylenol, hexyl-resorcinol, or sodium dodecyl sulfate stimulated further release of the chemokines from fibroblasts above that obtained with control media. Rabbit anti-interleukin-1alpha serum inhibited <em>keratinocyte</em>-conditioned media induction of chemokine release. We have shown a structure-cytotoxicity relationship for a series of phenols as well as an association of interleukin-1alpha release with a cytotoxic effect. We demonstrated a cytokine cascade amplification step by the actions of stimulated <em>keratinocyte</em> media on cultured dermal fibroblasts, identifying interleukin-1alpha as the principal initiator of chemokine synthesis.

Comparison of the protein expression patterns of proliferating normal primary human <em>keratinocytes</em> plated in serum-free medium (SFKM), supplemented with epidermal <em>growth</em> <em>factor</em> (EGF) and bovine pituitary extract (BPE), and similar cultures induced to differentiate by the addition of Dulbecco's modified Eagle medium (DMEM), containing 10% fetal calf serum (FCS), revealed several known and unknown polypeptides that are abnormally regulated in the differentiated cells. Upregulated proteins included keratins (keratins 6, 10/11, 14 and 16), members of the S100 protein family psoriasin, MRP8, MRP14 and S100c), actin-binding proteins (gelsolin and tropomyosin 9<em>2</em><em>2</em>0), annexins (annexins IV and VIII), hsp<em>2</em>8, the fatty acid binding protein 5 (FABP5), the squamous cell carcinoma (SCC) antigen, members of the 14-3-3 family, involucrin, E-cadherin, cystatin A, desmoglein and integrins alpha <em>2</em> and beta 1, as well as several proteins of as yet unknown identity. The highest upregulated proteins corresponded to psoriasin (1<em>2</em>4.0 times), MRP8 (4<em>2</em>.4 times), MRP14 (14.9 times), tropomyosin 9<em>2</em><em>2</em>0 (11.5 times), involucrin (11.1 times), and FABP5 (9.1 times). FABP5, hsp<em>2</em>8, and tropomyosin 9<em>2</em><em>2</em>0 were also highly upregulated in quiescent <em>keratinocytes</em> indicating that their increased levels in the differentiated cells may be due to loss of proliferative activity. Highly downregulated proteins included PAI-<em>2</em>, tropomyosins 9<em>2</em>13, 91<em>2</em>1 and 91<em>2</em><em>2</em>, keratin 5, calnexin, 14-3-3 beta and eta, nucleoside diphosphate kinase A, Rho GDIs, hsp60, hnRNPs H and C<em>2</em>, alpha-enolase, eIF-4D, thioredoxin, annexins III and V, moesin, nucleolar protein B<em>2</em>3, GST pi and PCNA/cyclin. Both the high expression of keratin 6 and 16--which are markers for an alternative pathway of <em>keratinocyte</em> differentiation--as well as the extremely high upregulation of some members of the S100 protein family indicate that the cells have differentiated via an abnormal pathway.

In this study we show that both cultured normal human epidermal cells (EC) and a human squamous cell carcinoma (SCC) cell line produce a thymocyte-activating <em>factor</em> (ETAF). EC-ETAF and SCC-ETAF both have a Mr of 15,000 and were eluted from chromatofocusing at the same isoelectric points of 7.<em>2</em>, 5.8, and 5.0. Both activities were maintained at alkaline pH and were destroyed at temperatures above 60 degrees C. In addition to stimulating thymocyte proliferation, human ETAF exhibited a variety of other pertinent biologic activities. Although EC-ETAF or SCC-ETAF by themselves exhibited no T-cell <em>growth</em> <em>factor</em> activity, both ETAF preparations enhanced Interleukin <em>2</em> production by cultured human peripheral blood lymphocytes when stimulated with polyclonal T-cells stimulants (Concanavalin A and phorbol myristate acetate). Human ETAF also was chemotactic for rabbit polymorphonuclear leukocytes and was directly mitogenic for cultured human dermal fibroblasts. Injection of human ETAF into C3H/HeJ mice, resulted in inducing serum amyloid A (SAA) production by murine hepatocytes. The thymocyte <em>growth</em>-enhancing activity, the fibroblast-stimulating activity, and the SAA-inducing capacity of ETAF all coeluted off AcA54 gel. These biologic as well as biochemical properties of human <em>keratinocyte</em>-derived ETAF are identical with those of human macrophage-derived Interleukin 1. The ability of <em>keratinocytes</em> to release an immunomodulating <em>factor</em> with such diverse consequences may play an important role in normal wound healing and in diseases involving epithelial tissues.

In patients undergoing radiation therapy in the thoracic region, ionizing radiation causes immediate damage to pulmonary endothelial and epithelial cells. We have recently shown that <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) protects against increases in permeability induced by hydrogen peroxide in human airway epithelial cells. Since radiation injury involves the production of oxygen free radicals, we tested the hypothesis that KGF would protect against radiation-induced increases in permeability. Two lines of human airway epithelial cells (Calu-3 and 16HBE14o-) were grown on collagen-coated polyester membranes (Transwell, Costar) and the permeability of the monolayers was determined by measuring the flux of tracers from the top chamber to the bottom chamber as a function of time. Changes in permeability were apparent 4 h after exposure. Increasing doses of radiation (<em>2</em>-10 Gy) stimulated significant increases in permeability compared with control monolayers (P < 0. 05, n=5-10) in Calu-3 and 16HBE14o- cells. KGF (50 ng/ml) alone reduced permeability significantly compared with controls, protected against increases in permeability with low doses of radiation and provided partial protection at higher doses. KGF also provided a significant effect in cells irradiated with 10 Gy (n=5, P < 0. 05) when given for the 4 h immediately after irradiation. The effects of KGF were sustained. After a full <em>2</em>4-h pretreatment with KGF, cells were incubated in medium without KGF for 8 or 1<em>2</em> h prior to 10 Gy irradiation. Both of these treatments significantly reduced permeability to albumin in sham-irradiated and irradiated cells (n=3, P < 0.05). To investigate the signal transduction pathways through which KGF mediates protection, permeability was measured in the presence of the protein kinase C (PKC) inhibitor, calphostin C, or the tyrosine kinase inhibitor, genistein. Inhibition of PKC blocked the decrease in basal tracer flux caused by KGF treatment in both cell types and removed the KGF-mediated protection against radiation. Incubation with genistein completely blocked the KGF-mediated decrease in the baseline tracer flux, as well as the ameliorating effect observed after irradiation. Rhodamine-phalloidin staining of the F-actin cytoskeleton showed disruption of the cytoskeleton with radiation exposure, increased density of F-actin filaments with KGF treatment, and resistance to disruption when cells were pretreated with KGF and exposed to radiation. Our results suggest that KGF regulates permeability in airway epithelium through a pathway mediated by PKC and tyrosine kinase that stabilizes the F-actin cytoskeleton.