BACKGROUND

Although the precise pathomechanism of psoriasis is still unknown, various cytokines and growth factors derived from T cells, dendritic cells or keratinocytes, are critically involved in this disease. There have been several studies determining the serum levels of cytokines in patients with psoriasis, but with conflicting results. The levels of various cytokines and growth factors were measured in the sera of patients with psoriasis and compared with those of healthy controls. The correlation with disease severity was also determined.

METHODS

Sera were collected from 122 patients with psoriasis and 78 healthy controls for ELISA analysis to evaluate the levels of cytokines and growth factors. The severity of psoriasis was determined by the Psoriasis Area and Severity Index (PASI).

RESULTS

Serum levels of tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-2, IL-6, IL-7, IL-8, IL-12, IL-17, IL-18 and vascular endothelial growth factor (VEGF) were significantly increased in patients with psoriasis compared with those of healthy controls. The serum levels of IL-2, soluble intercellular adhesion molecule-1, epidermal growth factor, hepatocyte growth factor and amphiregulin were not significantly different from those of healthy controls. Increased serum levels of TNF-alpha, IFN-gamma, IL-12, IL-17, IL-18 and VEGF correlated with PASI. Furthermore, these cytokine levels were decreased after psoriasis treatment. In contrast, serum levels of IL-10 were decreased in psoriasis and negatively correlated with PASI.

CONCLUSIONS

Serum levels of TNF-alpha, IFN-gamma, IL2, IL-6, IL-7, IL-8, IL-12, IL-17, IL-18 and VEGF were positively correlated and that of IL-10 was negatively correlated with PASI in Japanese patients with psoriasis. These parameters might be useful for determining the disease activity of psoriasis.

Human papillomaviruses (HPVs) cause benign and malignant tumors of the mucosal and cutaneous epithelium. The initial events regulating HPV infection impact the establishment of viral persistence, which is requisite for malignant progression of HPV-infected lesions. However, the precise mechanisms involved in HPV entry into host cells, including the cellular <em>factors</em> regulating virus uptake, are not clearly defined. We show that HPV16 exposure to human <em>keratinocytes</em> initiates epidermal <em>growth</em> <em>factor</em> receptor (EGFR)-dependent Src protein kinase activation that results in phosphorylation and extracellular translocation of annexin A<em>2</em> (AnxA<em>2</em>). HPV16 particles interact with AnxA<em>2</em> in association with S100A10 as a heterotetramer at the cell surface in a Ca(<em>2</em>+)-dependent manner, and the interaction appears to involve heparan-sulfonated proteoglycans. We show multiple lines of evidence that this interaction promotes virus uptake into host cells. An antibody to AnxA<em>2</em> prevents HPV16 internalization, whereas an antibody to S100A10 blocks infection at a late endosomal/lysosomal site. These results suggest that AnxA<em>2</em> and S100A10 have separate roles during HPV16 binding, entry, and trafficking. Our data additionally imply that AnxA<em>2</em> and S100A10 may be involved in regulating the intracellular trafficking of virus particles prior to nuclear delivery of the viral genome.

Vemurafenib (PLX403<em>2</em>), a selective inhibitor of Braf, has been approved by the US Food and Drug Administration for the treatment of unresectable or metastatic melanoma in patients with Braf(V600E) mutations. Many patients treated with vemurafenib initially display dramatic improvement, with decreases in both risk of death and tumor progression. Acquired resistance, however, rapidly arises in previously sensitive cells. We attempted to overcome this resistance by targeting the signal transducer and activator of transcription 3 (STAT3)-paired box homeotic gene 3 (PAX3)-signaling pathway, which is upregulated, owing to fibroblast <em>growth</em> <em>factor</em> <em>2</em> (FGF<em>2</em>) secretion or increased kinase activity, with the Braf(V600E) mutation. We found that activation of Stat3 or overexpression of PAX3 induced resistance to vemurafenib in melanoma cells. In addition, PAX3 or Stat3 silencing inhibited the <em>growth</em> of melanoma cells with acquired resistance to vemurafenib. Furthermore, treatment with the Stat3 inhibitor, WP1066, resulted in <em>growth</em> inhibition in both vemurafenib-sensitive and -resistant melanoma cells. Significantly, vemurafenib stimulation induced FGF<em>2</em> secretion from <em>keratinocytes</em> and fibroblasts, which might uncover, at least in part, the mechanisms underlying targeting Stat3-PAX3 signaling to overcome the acquired resistance to vemurafenib. Our results suggest that Stat3-targeted therapy is a new therapeutic strategy to overcome the acquired resistance to vemurafenib in the treatment of melanoma.

The epidermis (containing primarily <em>keratinocytes</em> and melanocytes) overlies the dermis (containing primarily fibroblasts) of human skin. We previously reported that dickkopf 1 (DKK1) secreted by fibroblasts in the dermis elicits the hypopigmented phenotype of palmoplantar skin due to suppression of melanocyte function and <em>growth</em> via the regulation of two important signaling <em>factors</em>, microphthalmia-associated transcription <em>factor</em> (MITF) and beta-catenin. We now report that treatment of <em>keratinocytes</em> with DKK1 increases their proliferation and decreases their uptake of melanin and that treatment of reconstructed skin with DKK1 induces a thicker and less pigmented epidermis. DNA microarray analysis revealed many genes regulated by DKK1, and several with critical expression patterns were validated by reverse transcriptase-polymerase chain reaction and Western blotting. DKK1 induced the expression of keratin 9 and alpha-Kelch-like ECT<em>2</em> interacting protein (alphaKLEIP) but down-regulated the expression of beta-catenin, glycogen synthase kinase 3beta, protein kinase C, and proteinase-activated receptor-<em>2</em> (PAR-<em>2</em>), which is consistent with the expression patterns of those proteins in human palmoplantar skin. Treatment of reconstructed skin with DKK1 reproduced the expression patterns of those key proteins observed in palmoplantar skin. These findings further elucidate why human skin is thicker and paler on the palms and soles than on the trunk through topographical and site-specific differences in the secretion of DKK1 by dermal fibroblasts that affects the overlying epidermis.

IL-6, which is also known as IFN-beta <em>2</em>, hybridoma <em>growth</em> <em>factor</em>, hepatocyte-stimulating <em>factor</em>, and B cell differentiation <em>factor</em>, mediates acute phase responses including fever, has lymphocyte-stimulating capacities, and antiviral activity. IL-6 is produced by monocytes, fibroblasts, certain lymphocytes, and various tumor cells. The present study demonstrates that this multifunctional cytokine is released also by normal human epidermal cells (EC) and human epidermoid carcinoma cell lines (A431, KB). Accordingly, supernatants derived from freshly isolated EC, long term <em>keratinocyte</em> cultures, A431, or KB cells stimulated the proliferation of a hybridoma <em>growth</em> <em>factor</em>/IL-6-dependent plasmacytoma cell line (B9). IL-6 constitutively was produced in the presence of serum proteins. The addition of IL-1 alpha, IL-1 beta, or the tumor promoter PMA significantly enhanced the synthesis and release of EC-derived IL-6 (EC-IL 6). Like monocyte or fibroblast-derived IL-6, EC-IL-6 exhibited Mr microheterogeneity within <em>2</em>1 and <em>2</em>8 kDa. Similarly in Western blotting experiments an antiserum directed against human rIFN-beta <em>2</em>/IL-6 detected the different Mr forms of EC-IL-6. Moreover, this antiserum was able to block the B9 cell <em>growth</em>-promoting capacity of EC-IL-6 strongly suggesting that this EC-derived mediator is closely related, if not identical with IL-6. This was further confirmed by Northern blot analysis detecting IL-6 specific mRNA both in long term cultured <em>keratinocytes</em> and A431 cells by hybridization with a cDNA fragment encoding for B cell differentiating <em>factor</em> <em>2</em>/IL-6. Therefore, in addition to the production of other cytokines as previously reported, EC and in particular <em>keratinocytes</em> also synthesize and release IL-6. This further supports the important regulatory role of the epidermis during the pathogenesis of inflammatory, autoimmune, and neoplastic diseases.

Hyaluronan, a major extracellular matrix molecule in the vital cell layers of skin epidermis, has been suggested to support proliferation and migration of <em>keratinocytes</em>, during challenges like wounding and inflammation. An organotypic <em>keratinocyte</em> culture originated from continuous rat epidermal <em>keratinocyte</em> cell line was subjected to the proliferative and antiproliferative <em>growth</em> <em>factors</em> epidermal <em>growth</em> <em>factor</em> and transforming <em>growth</em> <em>factor</em> beta, respectively, to study their influence on hyaluronan synthesis and epidermal morphology. Epidermal <em>growth</em> <em>factor</em> induced a 4-fold increase of epidermal hyaluronan concentration. This was associated with upregulation of the hyaluronan synthases Has<em>2</em> and Has3, and the hyaluronan receptor CD44. 5-Bromo-<em>2</em>'-deoxyuridine labeling, basal cell height, and the thickness of vital epidermis were increased, reflecting the hyperplastic effects of epidermal <em>growth</em> <em>factor</em>. The expression of keratin 10 and the maturation of filaggrin were inhibited, and epidermal permeability barrier became less efficient, indicating compromised terminal differentiation by epidermal <em>growth</em> <em>factor</em>. In contrast, transforming <em>growth</em> <em>factor</em> beta reduced the content of hyaluronan and the mRNA of Has<em>2</em> and Has3. At the same time, transforming <em>growth</em> <em>factor</em> beta suppressed <em>keratinocyte</em> proliferation and epidermal thickness, but retained intact differentiation. The results suggest that epidermal hyaluronan synthesis, controlled by epidermal <em>growth</em> <em>factor</em> and transforming <em>growth</em> <em>factor</em> beta through changes in the expression of Has<em>2</em> and Has3, correlates with epidermal proliferation, thickness, and differentiation.

The cellular members of the interleukin-10 (IL-10) cytokine family share sequence homology with IL-10, whereas their sites of expression and their functions are divergent. One of these <em>factors</em>, AK155 or IL-<em>2</em>6, was discovered because of its overexpression in human T lymphocytes after <em>growth</em> transformation by the simian rhadinovirus herpesvirus saimiri. In addition, the gene is transcribed in various types of primary and immortalized T-cells. Here we describe epithelial cells, namely colon carcinoma cells and <em>keratinocytes</em>, as targets of this T-cellular lymphokine. Purified recombinant IL-<em>2</em>6 induced the rapid phosphorylation of the signal transducer and activator of transcription <em>factors</em> 1 and 3. As a result, secretion of IL-10 and IL-8, as well as cell surface expression of CD54 were enhanced. Moreover, we show that the IL-<em>2</em>6 protein binds to heparin, is released from the cell surface, and can be functionally inhibited by heparin. The sensitivity to recombinant IL-<em>2</em>6 of various cell lines strictly correlated with the expression of the long chain of the IL-<em>2</em>0 receptor. Because blocking antibodies against either the short chain of the IL-10 receptor or the long chain of the IL-<em>2</em>0 receptor inhibited IL-<em>2</em>6-dependent signal transduction, and transient expression of these receptor chains induced IL-<em>2</em>6 responsivity in non-sensitive cells, we propose that the IL-<em>2</em>0 receptor 1 and IL-10 receptor <em>2</em> chains participate in forming the IL-<em>2</em>6 receptor. Targeting epithelial cells, the T-cell lymphokine IL-<em>2</em>6 is likely to play a role in local mechanisms of mucosal and cutaneous immunity.

Members of Snail family of transcription <em>factors</em> play an important role in oral cancer progression by inducing epithelial-mesenchymal transition, by promoting invasion and by increasing matrix metalloproteinase (MMP) expression. Although Snail (Snai1) is the best characterized and the most extensively studied member of this family, the role and regulation of Slug (Snai<em>2</em>) in oral cancer progression is less well understood. In this report, we show that transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1) increases Slug levels in tert-immortalized oral <em>keratinocytes</em> and in malignant oral squamous cell carcinoma (OSCC) cells. Inhibiting ERK1/<em>2</em> signaling, but not PI3-kinase signaling, blocked TGF-beta1-induced Slug expression in the malignant UMSCC1 cells. To further examine the role of Slug in OSCC progression, we generated UMSCC1 cells with inducible expression of Slug protein. Induction of Slug in UMSCC1 cells did not repress E-cadherin levels or regulate individual movement of UMSCC1 cells. Instead, Slug enhanced cohort migration and Matrigel invasion by UMSCC1 cells. Slug increased MMP-9 levels and MMP-9-specific siRNA blocked Slug-induced Matrigel invasion. Interestingly, Slug-specific siRNA attenuated TGF-beta1-induced MMP-9 expression and Matrigel invasion. These data demonstrate that TGF-beta1 increases Slug via ERK1/<em>2</em> signaling, and thereby contributes to OSCC progression.

The insulin-like <em>growth</em> <em>factor</em> 1 receptor (IGF-1R) is a multifunctional receptor that mediates signals for cell proliferation, differentiation, and survival. Genetic experiments showed that IGF-1R inactivation in skin results in a disrupted epidermis. However, because IGF-1R-null mice die at birth, it is difficult to study the effects of IGF-1R on skin. By using a combined approach of conditional gene ablation and a three-dimensional organotypic model, we demonstrate that IGF-1R-deficient skin cocultures show abnormal maturation and differentiation patterns. Furthermore, IGF-1R-null <em>keratinocytes</em> exhibit accelerated differentiation and decreased proliferation. Investigating the signaling pathway downstream of IGF-1R reveals that insulin receptor substrate <em>2</em> (IRS-<em>2</em>) overexpression compensates for the lack of IGF-1R, whereas IRS-1 overexpression does not. We also demonstrate that phosphatidylinositol 3-kinase and extracellular signal-regulated kinase 1 and <em>2</em> are involved in the regulation of skin <em>keratinocyte</em> differentiation and take some part in mediating the inhibitory signal of IGF-1R on differentiation. In addition, we show that mammalian target of rapamycin plays a specific role in mediating IGF-1R impedance of action on <em>keratinocyte</em> differentiation. In conclusion, these results reveal that IGF-1R plays an inhibitory role in the regulation of skin development and differentiation.

Treatment of normal human epidermal <em>keratinocytes</em> (NHEK) with interferon-gamma (IFN-gamma) causes a 9-fold increase in the level of cyclooxygenase-<em>2</em> (COX-<em>2</em>) mRNA expression. Nuclear run-off assays indicate that this induction is at least partly due to increased transcription. Activation of the epidermal <em>growth</em> <em>factor</em> receptor (EGFR) signaling pathway due to the enhanced transforming <em>growth</em> <em>factor</em> alpha (TGFalpha) expression plays an important role in the induction of COX-<em>2</em> by IFN-gamma. This is supported by the ability of TGFalpha to rapidly induce COX-<em>2</em> and the inhibition of the IFN-gamma-mediated COX-<em>2</em> mRNA induction by an EGFR antibody and EGFR-selective kinase inhibitors. Deletion and mutation analysis indicates the importance of the proximal cAMP-response element/ATF site in the transcriptional control of this gene by TGFalpha. The increase in COX-<em>2</em> mRNA by TGFalpha requires activation of both the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) pathways. Inhibition of p38 MAPK decreases the stability of COX-<em>2</em> mRNA, while inhibition of MAPK/ERK kinase (MEK) does not. These results suggest that the p38 MAPK signaling pathway controls COX-<em>2</em> at the level of mRNA stability, while the ERK signaling pathway regulates COX-<em>2</em> at the level of transcription. In contrast to NHEK, IFN-gamma and TGFalpha are not very effective in inducing TGFalpha or COX-<em>2</em> expression in several squamous carcinoma cell lines, indicating alterations in both IFN-gamma and TGFalpha response pathways.

The antitumor effect of curcumin (diferuloylmethane) is well established. However, there have been no unbiased studies to identify novel molecular targets of this compound. We therefore undertook a gene expression profiling study to identify novel targets of curcumin. A cDNA array comprised of 1<em>2</em>,6<em>2</em>5 probes was used to compare total RNA extracted from curcumin-treated and untreated MDA-1986 cells for differential gene expression. We identified <em>2</em>0<em>2</em> up-regulated mRNAs and 505 transcripts decreased>> or =<em>2</em>-fold. The proapoptotic activating transcription <em>factor</em> 3 (ATF3) was induced >4-fold. Two negative regulators of <em>growth</em> control [antagonizer of myc transcriptional activity (Mad) and p<em>2</em>7kip1] were induced 68- and 3-fold, respectively. Additionally, two dual-activity phosphatases (CL 100 and MKP-5), which inactivate the c-jun-NH<em>2</em>-kinases, showed augmented expression, coinciding with reduced expression of the upstream activators of c-jun-NH<em>2</em>-kinase (MEKK and MKK4). Of the repressed genes, the expression of Frizzled-1 (Wnt receptor) was most strongly attenuated (8-fold). Additionally, two genes implicated in <em>growth</em> control (K-sam, encoding the <em>keratinocyte</em> <em>growth</em> <em>factor</em> receptor, and HER3) as well as the E<em>2</em>F-5 transcription <em>factor</em>, which regulates genes controlling cell proliferation, also showed down-regulated expression. Considering its role in apoptosis, we determined the contribution of ATF3 to the antitumor effect of curcumin. Curcumin-treated MDA-1986 cells showed a rapid, dose-dependent increase in ATF3/mRNA protein. Moreover, expression of an exogenous ATF3 cDNA synergized with curcumin in inducing apoptosis. Thus, we have identified several putative, novel molecular targets of curcumin and showed that one, (ATF3) contributes to the proapoptotic effects of this compound.

In this study, we examined the impact of matrix metalloproteinases (MMP) on epithelialization, granulation tissue development, wound contraction, and alpha-smooth muscle actin (ASMA) expression during cutaneous wound repair through systemic administration of the synthetic broad-spectrum MMP inhibitor GM 6001 (N-[(<em>2</em>R)-<em>2</em>-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide). Four full-thickness excisional wounds (50 mm<em>2</em>) on the back of <em>2</em><em>2</em> young female Sprague-Dawley rats, 1<em>2</em> treated with GM 6001 100 mg/kg and 10 with vehicle, were allowed to heal by secondary intention. GM 6001-treated wounds were minimally resurfaced with neoepithelium, despite unaltered <em>keratinocyte</em> proliferation in wound edges, whereas control wounds were completely covered with 3-7 cell layers of parakeratinized epithelium on post-wounding day 7. Hydroxyproline concentration, a marker of collagen, and cell proliferation in granulation tissue did not differ significantly between GM 6001-treated and control groups. Impaired wound contraction (P < 0.01) was associated with a dramatic reduction of ASMA-positive myofibroblasts in granulation tissue of GM 6001 wounds. This was not due to GM6001 blocking transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1)-induced myofibroblast differentiation since GM 6001 did not inhibit TGF-beta1-induced ASMA expression and force generation in cultured rat dermal fibroblasts. The profound impairment of skin repair by the nonselective MMP inhibitor GM 6001 suggests that <em>keratinocyte</em> resurfacing, wound contraction, and granulation tissue organization are highly MMP-dependent processes.

Full-skin substitutes, epidermal substitutes, and dermal substitutes are currently being used to heal deep burns and chronic ulcers. In this study, we investigated which wound-healing mediators are released from these constructs and whether <em>keratinocyte</em>-fibroblast interactions are involved. Autologous skin substitutes were constructed from human <em>keratinocytes</em>, fibroblasts, and acellular donor dermis. Full-thickness skin was used to represent an autograft. Secretion of wound-healing mediators was investigated by means of protein array, enzyme-linked immunosorbent assay, neutralizing antibodies, and conditioned culture supernatants. Full-skin substitutes and autografts produce high amounts of inflammatory/angiogenic mediators (IL-6, CCL<em>2</em>, CXCL1, CXCL8, and sST<em>2</em>). Epidermal and dermal substitutes produced less of these proteins. Epidermal-derived proinflammatory cytokines interleukin-1alpha (IL-1alpha) and tumor necrosis <em>factor</em>-alpha (TNF-alpha) were found to mediate synergistically the secretion of these wound-healing mediators (with the exception of sST<em>2</em>) from fibroblasts in dermal substitutes. The secretion of proinflammatory cytokines (IL-1alpha, TNF-alpha), chemokine/mitogen (CCL5) and angiogenic <em>factor</em> (vascular endothelial <em>growth</em> <em>factor</em>) by epidermal substitutes and tissue remodeling <em>factors</em> (tissue inhibitor of metalloproteinase-<em>2</em>, hepatocyte <em>growth</em> <em>factor</em>) by dermal substitutes was not influenced by <em>keratinocyte</em>-fibroblast interactions. The full-skin substitute has a greater potential to stimulate wound healing than epidermal or dermal substitutes. Both epidermal-derived IL-1alpha and TNF-alpha are required to trigger the release of dermal-derived inflammatory/angiogenic mediators from skin substitutes.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) is a member of the heparin-binding fibroblast <em>growth</em> <em>factor</em> family (FGF-7) with a distinctive pattern of target-cell specificity. Studies performed in cell culture suggested that KGF was mitogenically active only on epithelial cells, albeit from a variety of tissues. In contrast, KGF was produced solely by cells of mesenchymal origin, leading to the hypothesis that it might function as a paracrine mediator of mesenchymal-epithelial communication. Biochemical analysis and molecular cloning established that the KGF receptor (KGFR) was a tyrosine kinase isoform encoded by the fgfr-<em>2</em> gene. Many detailed investigations of KGF and KGFR expression in whole tissue and cell lines largely substantiated the pattern initially perceived in vitro of mesenchymal and epithelial distribution, respectively. Moreover, functional assays in organ culture and in vivo and studies of KGF regulation by sex steroid hormones reinforced the idea that KGF acts predominantly on epithelial cells to elicit a variety of responses including proliferation, migration and morphogenesis.

Hyaluronan is an abundant and rapidly turned over matrix molecule between the vital cell layers of the epidermis and subject to large concentration changes associated with <em>keratinocyte</em> proliferation, migration, and differentiation induced by paracrine and endocrine <em>factors</em> like epidermal <em>growth</em> <em>factor</em> (EGF) and all-trans-retinoic acid (RA). We found that in REK cells EGF and all-trans-RA up-regulated hyaluronan synthase <em>2</em> (Has<em>2</em>) gene expression within <em>2</em> h 4-fold each and in HaCaT human immortal <em>keratinocytes</em> 8- and 33-fold, respectively. The first 10 kb of the human Has<em>2</em> promoter were scanned in silico and in vitro for potential response elements of signal transducer and activator of transcription (STAT) or RA receptor (RAR) proteins. We identified a STAT-response element in the proximal promoter region and confirmed its functionality in response to EGF by chromatin immunoprecipitation (ChIP) assays. Direct in vitro binding of RARs to four RARE candidates within the Has<em>2</em> promoter could not be observed at stringent gel shift conditions, but reporter gene assays demonstrated functionality of a complex of two of these RAREs located approximately 1<em>2</em>00 bp upstream of the transcription start site. Moreover, ChIP assays using antibodies against nine nuclear proteins monitored all-trans-RA-dependent binding of RAR, retinoid X receptor, mediator protein, and RNA polymerase II and also histone 4 acetylation to a promoter region containing the complex RARE. Taken together, the human Has<em>2</em> gene is a potent primary EGF and all-trans-RA responding gene with a complex regulation.

The epidermis, our first line of defense from ultraviolet (UV) light, bears the majority of photodamage, which results in skin thinning, wrinkling, keratosis, and malignancy. Hypothesizing that skin has specific mechanisms to protect itself and the organism from UV damage, we used DNA arrays to follow UV-caused gene expression changes in epidermal <em>keratinocytes</em>. Of the 6,800 genes examined, UV regulates the expression of at least 198. Three waves of changes in gene expression can be distinguished, 0.5-<em>2</em>, 4-8, and 16-<em>2</em>4 h after illumination. The first contains transcription <em>factors</em>, signal transducing, and cytoskeletal proteins that change cell phenotype from a normal, fast-<em>growing</em> cell to an activated, paused cell. The second contains secreted <em>growth</em> <em>factors</em>, cytokines, and chemokines; <em>keratinocytes</em>, having changed their own physiology, alert the surrounding tissues to the UV damage. The third wave contains components of the cornified envelope, as <em>keratinocytes</em> enhance the epidermal protective covering and, simultaneously, terminally differentiate and die, removing a carcinogenic threat. UV also induces the expression of mitochondrial proteins that provide additional energy, and the enzymes that synthesize raw materials for DNA repair. Using a novel skin organ culture model, we demonstrated that the UV-induced changes detected in <em>keratinocyte</em> cultures also occur in human epidermis in vivo.

Using an expression cloning strategy, we isolated a cDNA encoding a human protein-tyrosine-phosphatase. Bacteria expressing the kinase domain of the <em>keratinocyte</em> <em>growth</em> <em>factor</em> receptor (bek/fibroblast <em>growth</em> <em>factor</em> receptor <em>2</em>) were infected with a fibroblast cDNA library in a phagemid prokaryotic expression vector and screened with a monoclonal anti-phosphotyrosine antibody. Among several clones showing decreased anti-phosphotyrosine recognition, one displayed phosphatase activity toward the kinase in vitro. The 4.1-kilobase cDNA encoded a deduced protein of 185 amino acids with limited sequence similarity to the vaccinia virus phosphatase VH1. The purified recombinant protein dephosphorylated several activated <em>growth</em> <em>factor</em> receptors, as well as serine-phosphorylated casein, in vitro. Both serine and tyrosine phosphatase activities were completely abolished by mutagenesis of a single cysteine residue conserved in VH1 and the VH1-related (VHR) human protein. These properties suggest that VHR is capable of regulating intracellular events mediated by both tyrosine and serine phosphorylation.

Vascular endothelial <em>growth</em> <em>factor</em> (VEGF) plays an important role in normal and pathological angiogenesis. VEGF receptors (VEGFRs, including VEGFR-1, VEGFR-<em>2</em>, and VEGFR-3) and neuropilins (NRPs, including NRP-1 and NRP-<em>2</em>) are high-affinity receptors for VEGF and are typically considered to be specific for endothelial cells. Here we showed expression of VEGFRs and NRPs on cultured epidermal <em>keratinocytes</em> at both mRNA and protein levels. We further localized these receptors by immunofluorescence (IF) staining in the epidermis of surgical skin specimens. We found positive staining for VEGFRs and NRPs in all layers of the epidermis except for the stratum corneum. VEGFR-1 and VEGFR-<em>2</em> are primarily expressed on the cytoplasmic membrane of basal cells and the adjacent spinosum <em>keratinocytes</em>. All layers of the epidermis except for the horny cell layer demonstrated a uniform pattern of VEGFR-3, NRP-1, and NRP-<em>2</em>. Sections staining for NRP-1 and NRP-<em>2</em> also showed diffuse intense fluorescence and were localized to the cell membrane and cytoplasm of <em>keratinocytes</em>. In another panel of experiments, <em>keratinocytes</em> were treated with different concentrations of VEGF, with or without VEGFR-<em>2</em> neutralizing antibody in culture. VEGF enhanced the proliferation and migration of <em>keratinocytes</em>, and these effects were partially inhibited by pretreatment with VEGFR-<em>2</em> neutralizing antibody. Adhesion of <em>keratinocytes</em> to type IV collagen-coated culture plates was decreased by VEGF treatment, but this reduction could be completely reversed by pretreatment with VEGFR-<em>2</em> neutralizing antibody. Taken together, our results suggest that the expression of VEGFRs and NRPs on <em>keratinocytes</em> may constitute important regulators for its activity and may possibly be responsible for the autocrine signaling in the epidermis.

We isolated a clone encoding a protein from a human lens epithelial cell (LEC) cDNA library with antibody (Ab) from a cataract patient and named it "lens epithelium-derived <em>growth</em> <em>factor</em>" (LEDGF). LEDGF is found to be identical to p75, a coactivator of both transcription (1) and pre-mRNA splicing (<em>2</em>). In serum-free medium LEDGF stimulated <em>growth</em> of LECs, cos7 cells, skin fibroblasts, and <em>keratinocytes</em>, and prolonged cell survival. Without LEDGF, the aforementioned cells did not survive. Also in serum-free medium, Ab to LEDGF neutralizing LEDGF blocked cell <em>growth</em> and caused cell death. Thus, LEDGF, a regulatory <em>factor</em>, may play an important role for <em>growth</em> and survival of a wide range of cell types.

<em>Keratinocyte</em> migration is critical to reepithelialization during wound repair. The motility response is promoted by <em>growth</em> <em>factors</em>, cytokines, and cytokines produced in the wound bed, including those that activate the epidermal <em>growth</em> <em>factor</em> (EGF) receptor. The Alu-Leu-Arg-negative CXC chemokine interferon-inducible protein 9 (IP-9; also known as CXCL11, I-TAC, beta-R1, and H-174) is produced by <em>keratinocytes</em> in response to injury. As <em>keratinocytes</em> also express the receptor, CXCR3, this prompted us to examine the role and molecular mechanism by which IP-9 regulates <em>keratinocyte</em> motility. Unexpectedly, as CXCR3 liganding blocks <em>growth</em> <em>factor</em>-induced motility in fibroblasts, IP-9 alone promoted motility in undifferentiated <em>keratinocytes</em> (37 +/- 6% of the level of the highly motogenic EGF) as determined in a two-dimensional in vitro wound healing assay. IP-9 even enhanced EGF-induced motility in undifferentiated <em>keratinocytes</em> (116 +/- 5%; P < 0.05 compared to EGF alone), suggesting two separate mechanisms of action. IP-9-increased motility and -decreased adhesiveness required the intracellular protease calpain. The increases in both motility and calpain activity by IP-9 were blocked by pharmacological and molecular inhibition of phospholipase C-beta3 and chelation of calcium, which prevented an intracellular calcium flux. Molecular downregulation or RNA interference-mediated depletion of mu-calpain (calpain 1) but not M-calpain (calpain <em>2</em>) blocked IP-9-induced calpain activation and motility. In accord with elimination of IP-9-induced de-adhesion, RNA interference-mediated depletion of calpain 1 but not calpain <em>2</em> prevented cleavage of the focal adhesion component focal adhesion kinase and disassembly of vinculin aggregates. In comparison, EGF-induced motility of the same undifferentiated <em>keratinocytes</em> requires the previously described extracellular signal-regulated kinase to the M-calpain pathway. These data demonstrate that while both EGF- and IP-9-induced motility in <em>keratinocytes</em> requires calpain activity, the isoform of calpain triggered depends on the nature of the receptor for the particular ligand. Interestingly, physiological nonapoptotic calcium fluxes were capable of activating mu-calpain, implying that the calcium requirement of mu-calpain for activation is attained during cell signaling. This is also the first demonstration of differential activation of the two ubiquitous calpain isoforms in the same cell by different signals.

Anticancer drugs stimulate apoptosis in the hair follicles (HF) and cause hair loss, the most common side effect of chemotherapy. In a mouse model for chemotherapy-induced hair loss, we demonstrate that p53 is essential for this process: in contrast to wild-type mice, p53-deficient mice show neither hair loss nor apoptosis in the HF <em>keratinocytes</em> that maintained active proliferation after cyclophosphamide treatment. HF in p53 mutants are characterized by down-regulation of Fas and insulin-like <em>growth</em> <em>factor</em>-binding protein 3 and by increased expression of Bcl-<em>2</em>. These observations indicate that local pharmacological inhibition of p53 may be useful to prevent chemotherapy-associated hair loss.

The expression of an oncogenic rasHa gene in epidermal <em>keratinocytes</em> stimulates the tyrosine phosphorylation of protein kinase C delta and inhibits its enzymatic activity (Denning, M. F., Dlugosz, A. A., Howett, M. K., and Yuspa, S. H. (1993) J. Biol. Chem. <em>2</em>68, <em>2</em>6079-<em>2</em>6081). <em>Keratinocytes</em> expressing an activated rasHa gene secrete transforming <em>growth</em> <em>factor</em> alpha (TGFalpha) and have an altered response to differentiation signals involving protein kinase C (PKC). Because the neoplastic phenotype of v-rasHa expressing <em>keratinocytes</em> can be partially mimicked in vitro by chronic treatment with TGF alpha and the G protein activator aluminum fluoride (AlF4-), we determined if TGF alpha or AlF4- could induce tyrosine phosphorylation of PKCdelta. Treatment of primary <em>keratinocyte</em> cultures for 4 days with TGFalpha induced tyrosine phosphorylation of PKCdelta, whereas AlF4- only slightly stimulated PKCdelta tyrosine phosphorylation. The PKCdelta that was tyrosine-phosphorylated in response to TGFalpha had reduced activity compared with the nontyrosine-phosphorylated PKCdelta. Treatment of <em>keratinocytes</em> expressing a normal epidermal <em>growth</em> <em>factor</em> receptor (EGFR) with TGFalpha or epidermal <em>growth</em> <em>factor</em> for 5 min induced PKCdelta tyrosine phosphorylation. This acute epidermal <em>growth</em> <em>factor</em> treatment did not induce tyrosine phosphorylation of PKCdelta in <em>keratinocytes</em> isolated from waved-<em>2</em> mice that have a defective epidermal <em>growth</em> <em>factor</em> receptor. In addition, the level of PKCdelta tyrosine phosphorylation in v-rasHa-transduced <em>keratinocytes</em> from EGFR null mice was substantially lower than in v-rasHa transduced wild type cells, suggesting that activation of the EGFR is important for PKC delta tyrosine phosphorylation in ras transformation. However, purified EGFR did not phosphorylate recombinant PKC delta in vitro, whereas members of the Src family (c-Src, c-Fyn) and membrane preparations from <em>keratinocytes</em> did. Furthermore, clearing c-Src or c-Fyn from <em>keratinocyte</em> membrane lysates decreased PKCdelta tyrosine phosphorylation, and c-Src and c-Fyn isolated from <em>keratinocytes</em> treated with TGFalpha had increased kinase activity. Acute or chronic treatment with TGFalpha did not induce significant PKCdelta translocation in contrast to the phorbol ester 1<em>2</em>-O-tetradecanoylphorbol-13-acetate, which induced both translocation and tyrosine phosphorylation of PKCdelta. This suggests that TGFalpha-induced tyrosine phosphorylation of PKC delta results from the activation of a tyrosine kinase rather than physical association of PKCdelta with a membrane-anchored tyrosine kinase. Taken together, these results indicate that PKCdelta activity is inhibited by tyrosine phosphorylation in response to EGFR-mediated signaling and activation of a member of the Src kinase family may be the proximal tyrosine kinase acting on PKCdelta in <em>keratinocytes</em>.

We have previously shown that hydrogen peroxide is an important mediator of ultraviolet B induced phosphorylation of the epidermal <em>growth</em> <em>factor</em> receptor in human <em>keratinocytes</em>. Here we demonstrate that physiologic doses of ultraviolet B and hydrogen peroxide stimulate activation of two related but distinct mitogen-activated protein kinase pathways: extracellular regulated kinase 1 and <em>2</em> (ERK1/<em>2</em>), as well as p38, the mammalian homolog of HOG1 in yeast which is a major kinase for a recently identified stress-induced signaling pathway. The time-dependent activation of ERK1/<em>2</em> and p38 are distinct, and ultraviolet B-induced ERK1/<em>2</em> activation is downregulated more rapidly than p38. Using dihydrorhodamine or Amplex as specific fluorescent dye probes, we show that ultraviolet B-induced peroxides can be inhibited by ascorbic acid. Ascorbic acid strongly blocks ERK1/<em>2</em> and p38 activation by ultraviolet B and hydrogen peroxide whereas pyrrolidine dithiocarbamate and butyl hydroxyanisole are less effective. Pyrrolidine dithiocarbamate was unable to inhibit ultraviolet B-induced p38 activation. Cell death was increased after ultraviolet B when ERK1/<em>2</em> activation was attenuated by the specific inhibitor PD098059. The distinct time courses and extents of activation and inhibition of ERK1/<em>2</em> and p38 indicate that these pathways are separate and regulated independently in <em>keratinocytes</em>. Specific types of reactive oxygen species induced by ultraviolet B as well as selective activation or inhibition of specific phosphatases may mediate these responses in <em>keratinocytes</em>. These findings demonstrate that reactive oxygen species are important multifunctional mediators of ultraviolet B-induced ERK1/<em>2</em> and p38 signaling transduction pathways and suggest that ERK1/<em>2</em> may play an important part in protecting <em>keratinocytes</em> from cell death following oxidative stress.

Betel quid (BQ) chewing is an etiologic <em>factor</em> of oral cancer and submucus fibrosis (OSF). <em>Keratinocyte</em> inflammation is crucial for the pathogenesis of cancer and tissue fibrosis. We found that areca nut (AN) extract (100-400 micro g/ml) induced PGE<em>2</em> production by KB cells by <em>2</em>.34- to <em>2</em>3.1-fold and also TNF-alpha production by gingival <em>keratinocytes</em> (GK). Arecoline (0.<em>2</em>-1.<em>2</em> mM) elevated PGE<em>2</em> production by KB cells by <em>2</em>.5- to 6.1-fold. AN extract (<em>2</em>00-400 micro g/ml) also induced IL-6 production by GK (7.5- to 8.4-fold) and KB cells. In contrast, arecoline (0.1-1.<em>2</em> mM) suppressed IL-6 production by GK and KB cells, with 4<em>2</em>-81 and 41-63% inhibition, respectively. A 48 h exposure of GK to 800-1<em>2</em>00 micro g/ml AN extract led to 37-69% cell death. Arecoline cytotoxicity to GK was noted at concentrations of 0.8-1.<em>2</em> mM, which led to <em>2</em>8-38% cell death. AN extract (400-800 micro g/ml) induced Cox-<em>2</em> and IL-6 mRNA expression and also COX-<em>2</em> protein production by KB cells. IL-6 (5-100 ng/ml) suppressed GK <em>growth</em> by <em>2</em>0-33%, but enhanced oral fibroblast (OMF) and KB cell <em>growth</em>. PGE<em>2</em> (0.05-5 micro g/ml) and anti-IL-6 antibody (ab) (50-1000 ng/ml) showed little effect on GK and KB cell <em>growth</em>. Incubation of GK and KB cells with aspirin, anti-IL-6 ab and anti-TNF-alpha ab showed little effect on arecoline- and AN-induced cytotoxicity, cell cycle arrest and apoptosis. Exposure to anti-TNF-alpha ab slightly affected arecoline- and AN-modulated PGE<em>2</em> and IL-6 production by GK and KB cells. Arecoline- and AN-conditioned medium decreased phytohemagglutinin-mediated CD4+ and CD8+ T cell activation. These results indicate that BQ chewing contributes to the pathogenesis of cancer and OSF by impairing T cell activation and by induction of PGE<em>2</em>, TNF-alpha and IL-6 production, which affect oral mucosal inflammation and <em>growth</em> of OMF and oral epithelial cells.