Primary Cultures Of Human Keratinocytes Research Articles

Purinergic receptors are part of a functional signaling system for proliferation and differentiation of human epidermal keratinocytes.

We investigated the expression of P2X5, P2X7, P2Y1 and P2Y2 receptor subtypes in normal human epidermis and in relation to markers of proliferation (PCNA and Ki-67), keratinocyte differentiation (cytokeratin K10 and involucrin) and markers of apoptosis (TUNEL and anticaspase-3). Using immunohistochemistry, we showed that each of the four receptors was expressed in a spatially distinct zone of the epidermis, suggesting different functional roles for these receptors. Functional studies were performed on primary cultures of human keratinocytes and on explanted rat skin, where different P2 receptor subtype agonists and antagonists were applied to cultured keratinocytes or injected subcutaneously into the skin, respectively. An increase in cell number was caused by low doses of the nonspecific P2 receptor agonist ATP, the P2Y2 receptor agonist UTP (p<0.001), and the P2Y1 receptor agonist 2MeSADP (p<0.05). There was a significant decrease in cell number as a result of treatment with the P2X5 receptor agonist ATPgammaS (p<0.001) and the P2X7 receptor agonist BzATP (p<0.001). Suramin caused a significant block in the effect of 100 microm ATP (p<0.01) and 1000 microm ATP (p<0.001) on cell number. These results imply that different purinergic receptors have different functional roles in the human epidermis with P2Y1 and P2Y2 receptors controlling proliferation, while P2X5 and P2X7 receptors control early differentiation, terminal differentiation and death of keratinocytes, respectively.

Activation of adenovirus early promoters and lytic phase in differentiated strata of organotypic cultures of human keratinocytes.

Human oncolytic adenoviruses have been used in clinical trials targeting cancers of epithelial origin. To gain a better understanding of the infectious cycle of adenovirus in normal human squamous tissues, we examined the viral infection process in organotypic cultures of primary human keratinocytes. We show that for the infection to occur, wounding of the epithelium is required. In addition, infection appears to initiate at the basal or parabasal cells that express the high-affinity coxsackievirus-adenovirus receptor, CAR, whereas the productive phase takes place in differentiated cells. This is due, at least in part, to the differentiation-dependent activation of the E1A and E2A early promoters and E4 promoters. We also show that adenovirus infection triggers a response mediated by the abnormal accumulation of cyclin E and p21cip1 proteins similar to the one previously observed in human papillomavirus-infected tissues. However, the virus seems to be able to overcome it, at least partially.

Combination of vitamin D metabolites with selective inhibitors of vitamin D metabolism.

1alpha,25(OH)2D3 exerts antiproliferative, differentiating effects on many cell types, including cancer tissues. In most of its target cells, levels of 1alpha,25(OH)2D3 are regulated by local synthesis via CYP27B and metabolism via CYP24. Rapidly induced by vitamin D, CYP24 repeatedly hydroxylates the vitamin D side chain and ultimately terminates hormonal activity. Aiming at increased hormone levels, lifetime and function, numerous vitamin D analogs have been synthesized with structural modifications, which impede oxidation of the vitamin D side chain. Our group followed a different strategy, namely, blocking 1,25(OH)2D3 metabolism with inhibitors of CYP24. As appropriate inhibitors, we exploited compounds termed azoles, which directly bind to the heme iron of the CYPs via an azole nitrogen and to other parts of the substrate site. We synthesized some 400 azoles and tested their potential to selectively inhibit CYP24, but not hormone synthesis by the related CYP27B. Using primary human keratinocyte cultures as the source of CYP24 and CYP27, we discovered some 50 inhibitors of CYP24 with IC50 values in the nanomole range and selectivities up to 60-fold. As the first representative of selective CYP24 inhibitors, VID400 underwent preclinical development. In human keratinocytes, VID400 stabilized levels of endogenously produced 1alpha,25(OH)2D3, and thereby strongly amplified and prolonged expression of CYP24, a surrogate marker of hormonal function. In parallel, antiproliferative activity showed up at 100-fold or more lower concentrations of 1alpha,25(OH)2D3. This data suggests that CYP24 inhibitors could become attractive drugs in antiproliferative therapy, used as single entities to increase or extend endogenous hormone function or in combination with low doses of potent analogs. Moreover, we used selective inhibitors as valuable tools to (a) elucidate regulatory mechanisms of vitamin D synthesis and metabolism, (b) determine intrinsic activities of the otherwise highly transient vitamin D metabolites and (c) model the active sites of CYP24 and CYP27B.

The Promoter of the Human Proliferating Cell Nuclear Antigen Gene Is Not Sufficient for Cell Cycle-dependent Regulation in Organotypic Cultures of Keratinocytes

The proliferating cell nuclear antigen (PCNA) is essential for DNA replication of mammalian cells and their small DNA tumor viruses. The mechanism of the cell cycle-dependent regulation of the human PCNA promoter is not clear despite extensive investigations. In this report, we employed organotypic cultures of primary human keratinocytes, which closely resemble native skin comprising both proliferating and postmitotic, differentiated cells, to examine the cell cycle-dependent regulation of the human PCNA gene (hPCNA) in the absence or presence of the human papillomavirus type 18 (HPV-18) E7 protein. HPV-18 E7 promotes S phase re-entry in post-mitotic differentiated keratinocytes by abrogating the transcription repression of E2F transcription factors by the retinoblastoma susceptibility protein, pRb. We demonstrated that E7 reactivated the transcription of the endogenous hPCNA in differentiated keratinocytes. In contrast, with or without E7, the expression of a transduced hPCNA promoter-driven reporter did not correlate with that of the endogenous hPCNA gene in either proliferating or differentiated cells. Moreover, in Chinese hamster ovary and L-cells, HPV E7 and the adenovirus E1A protein repressed the transduced hPCNA promoter, but both activated an extended promoter construct spanning the first intron. Mutations of two E2F sites in the intron reduced the basal activity and abolished the response to E7 or E1A. Promoter repression or activation required the CR2 domain of E7 and, to a lesser extent, CR1 as well. However, in organotypic cultures, this extended promoter construct failed to recapitulate the cell cycle-dependent regulation of the endogenous hPCNA gene. Only when a full-length Myc-tagged hPCNA spanning the 5' promoter and all exons and introns was used was the native pattern of expression largely restored, indicative of the complexity of its regulation.

Alternative fates of keratinocytes transduced by human papillomavirus type 18 E7 during squamous differentiation.

The human papillomavirus type 18 (HPV-18) E7 protein promotes S-phase reentry in postmitotic, differentiated keratinocytes in squamous epithelium to facilitate vegetative viral DNA amplification. To examine the nature and fate of the differentiated cells that reenter S phase, organotypic cultures of primary human keratinocytes transduced with HPV-18 E7 were pulse-chase-pulse-labeled with (3)H-thymidine ((3)H-TdR) and bromodeoxyuridine (BrdU). The kinetics of the appearance of doubly labeled suprabasal cells demonstrate that E7 expression did not promote prolonged S phase. Rather, there was a considerable lag before a small percentage of the cells reentered another round of S phase. Fluorescence in situ hybridization analysis, indeed, revealed a small fraction of the cells with more than 4n chromosomes in the differentiated strata. Differentiated cells positive for (3)H-TdR, BrdU, or both often had enlarged nuclei or were binucleated. These results suggest that S phase is not followed by cell division, although nuclear division may occur. Interestingly, a significant fraction of differentiated cells that entered S phase subsequently accumulated p27kip1 protein with a kinetics preceding the accumulation of cyclin E. We conclude that E7-transduced, differentiated keratinocytes that enter S phase have two alternative fates: (i) a low percentage of cells undergoes endoreduplication, achieving higher than 4n ploidy, and (ii) a high percentage of cells accumulates the p27kip1, cyclin E, and p21cip1 proteins, resulting in arrest and preventing further S-phase reentry.

P21cip1 Degradation in differentiated keratinocytes is abrogated by costabilization with cyclin E induced by human papillomavirus E7.

The human papillomavirus (HPV) E7 protein promotes S-phase reentry in a fraction of postmitotic, differentiated keratinocytes. Here we report that these cells contain an inherent mechanism that opposes E7-induced DNA replication. In organotypic raft cultures of primary human keratinocytes, neither cyclin E nor p21cip1 is detectable in situ. However, E7-transduced differentiated cells not in S phase accumulate abundant cyclin E and p21cip1. We show that normally p21cip1 protein is rapidly degraded by proteasomes. In the presence of E7 or E6/E7, p21cip1, cyclin E, and cyclin E2 proteins were all up-regulated. The accumulation of p21cip1 protein is a posttranscriptional event, and ectopic cyclin E expression was sufficient to trigger it. In constract, cdk2 and p27kip1 were abundant in normal differentiated cells and were not significantly affected by E7. Cyclin E, cdk2, and p21cip1 or p27kip1 formed complexes, and relatively little kinase activity was found associated with cyclin E or cdk2. In patient papillomas and E7 raft cultures, all p27kip1-positive cells were negative for bromodeoxyuridine (BrdU) incorporation, but only some also contained cyclin E and p21cip1. In contrast, all cyclin E-positive cells also contained p27kip1. When the expression of p21cip1 was reduced by rottlerin, a PKC delta inhibitor, p27kip1- and BrdU-positive cells remained unchanged. These observations show that high levels of endogenous p27kip1 can prevent E7-induced S-phase reentry. This inhibition then leads to the stabilization of cyclin E and p21cip1. Since efficient initiation of viral DNA replication requires cyclin E and cdk2, its inhibition accounts for heterogeneous viral activities in productively infected lesions.

Fibroblast Growth Factor 10 Induces Proliferation and Differentiation of Human Primary Cultured Keratinocytes

Fibroblast growth factor 10 is a novel member of the fibroblast growth factor family, which is involved in morphogenesis and epithelial proliferation. It is highly homologous to the keratinocyte growth factor (or fibroblast growth factor 7), a key mediator of keratinocyte growth and differentiation. Both fibroblast growth factor 10 and keratinocyte growth factor bind with high affinity to the tyrosine kinase keratinocyte growth factor receptor. Here we analyzed the effect of fibroblast growth factor 10 on primary cultures of human keratinocytes, grown in chemically defined medium, and we compared the proliferative and differentiative cell responses to fibroblast growth factor 10 with those induced by keratinocyte growth factor and epidermal growth factor. Cell counting, 5-bromo-2'-deoxyuridine incorporation, and western blot analysis showed that fibroblast growth factor 10, similarly to keratinocyte growth factor, not only is a potent mitogen for human keratinocytes, but also promotes the expression of both early differentiation markers K1 and K10 and late differentiation marker filaggrin in response to the Ca2+ signal, and seems to sustain the proliferative activity in suprabasal stratified cells. Immunoprecipitation/western blot analysis revealed that fibroblast growth factor 10, similarly to keratinocyte growth factor, is able to induce tyrosine phosphorylation of keratinocyte growth factor receptor and of cellular substrates such as PLCgamma.

Selective inhibition of vitamin D hydroxylases in human keratinocytes

Human keratinocytes convert 25(OH)D 3 to hormonally active 1α,25(OH) 2D 3 and respond to its antiproliferative/prodifferentiating action in vitro and in vivo. Levels and activity of 1α,25(OH) 2D 3 are short-lived. 1α,25(OH) 2D 3 induces 24-hydroxylase (CYP24) that rapidly metabolizes the hormone, yielding a cascade of side-chain oxidized products and this eventually results in the loss of activity. Aiming at stabilizing the levels of active hormone, we have searched for potent, selective inhibitors of CYP24. Selective inhibition was crucial in order to avoid impairment of 1α,25(OH) 2D 3 synthesis, catalyzed by 1α-hydroxylase - a related member of cytochrome P-450 (CYP) superfamily. We describe here the testing protocol, using primary human keratinocyte cultures as an appropriate source of CYP24 and 1α-hydroxylase, 3H-25(OH)D 3 (at physiological concentrations) as substrate and sensitive HPLC techniques to analyze the complex metabolite profiles. Four hundred potential inhibitors were screened by this method; most of them were synthesized in our laboratory. These compounds (entitled azoles) were capable of direct binding to the heme iron and of additional interactions with other parts of the enzyme. In this paper, we present VID400 and SDZ 89–443, as first examples of powerful selective CYP24 inhibitors. As anticipated, these compounds increased the levels of 1α-hydroxylated products generated from 3H-25(OH)D 3 and extended their lifetime. Importantly, blocking of 24-hydroxylation led to a switch in metabolism, namely to preferential conversion of 1α,25(OH) 2D 3 to 1α,25(OH) 2-3epi-D 3. As spin-off from our program, selective inhibitors of 1α-hydroxylase were also found (e.g. SDZ 88–357). Using 3H-25(OH)D 3 as substrate in the absence of SDZ 88–357, CYP24 showed high preference for freshly generated 1α-hydroxylated metabolites over abundant 25(OH)D 3. In the presence of SDZ 88–357, we noticed a great increase in 24-hydroxylation of 3H-25(OH)D 3. Besides their use as valuable tools in elucidating regulatory mechanisms, inhibitors of VD hydroxylases may give rise to novel therapeutic strategies, especially in defects of cell growth and differentiation.

1α,25-Dihydroxy-3-epi-vitamin D 3 a physiological metabolite of 1α,25-dihydroxyvitamin D 3: Its production and metabolism in primary human keratinocytes

Recent studies of metabolism using pharmacological substrate concentrations of 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2D 3] in several tissues including primary cultures of human keratinocytes, bovine parathyroid cells and bone cells led to the identification of 1α,25-dihydroxy-3-epi-vitamin D 3 [1α,25(OH) 2-3-epi-D 3] as a major natural metabolite of 1α,25(OH) 2D 3. In the present study, we demonstrate that human keratinocytes incubated with 25-hydroxy[26,27- 3H] vitamin D 3 produce 1α,25(OH) 2-3-epi-D 3 along with 1α,25(OH) 2D 3. The production of 1α,25(OH) 2-3-epi-D 3 is also identified in human keratinocytes incubated with physiological substrate concentrations of 1α,25(OH) 2D 3. Unlike 24-hydroxylase, the major enzyme involved in the further metabolism of 1α,25(OH) 2D 3 in human keratinocytes, the enzyme(s) responsible for the production of 1α,25(OH) 2-3-epi-D 3 is constitutive and is not inhibited by ketoconazole. It is also noted that 1α,25(OH) 2-3-epi-D 3 is further metabolised in human keratinocytes into several as yet unidentified metabolites, the production of which is inhibited to a great extent by SDZ 89-443, an inhibitor of 24-hydroxylase. This finding indicates that the 24-hydroxylase like in the case of 1α,25(OH) 2D 3, also plays a major role in the metabolism of 1α,25(OH) 2-3-epi-D 3. The results obtained from the metabolism studies performed in parallel among 25OHD 3, 1α,25(OH) 2D 3 and 1α,25(OH) 2-3-epi-D 3 indicate that 1α,25(OH) 2-3-epi-D 3 and its metabolites exhibit higher metabolic stability. In summary, we demonstrate for the first time that 1α,25(OH) 2-3-epi-D 3 is a physiological metabolite of 1α,25(OH) 2D 3 in human keratinocytes. Also, 1α,25(OH) 2-3-epi-D 3 is further metabolised in human keratinocytes mainly through the activity of 24-hydroxylase. Furthermore, our finding of the relative metabolic stability of 1α,25(OH) 2-3-epi-D 3 and especially its metabolites when compared to 1α,25(OH) 2D 3 and its metabolites provides an important explanation for its previously observed potent inhibitory effect on keratinocyte growth in spite of its low affinity to vitamin D receptor.

Evidence for Involvement of the Epidermal Platelet-Activating Factor Receptor in Ultraviolet-B-Radiation-Induced Interleukin-8 Production

Ultraviolet B radiation has been shown to generate cutaneous inflammation in part through inducing oxidative stress and cytokine production in human keratinocytes. Amongst the proinflammatory cytokines synthesized in response to ultraviolet B radiation is the potent chemoattractant interleukin-8. Though the lipid mediator platelet-activating factor (PAF) is synthesized in response to oxidative stress, and keratinocytes express PAF receptors linked to cytokine biosynthesis, it is not known whether PAF is involved in ultraviolet-B-induced epidermal cell cytokine production. These studies examined the role of the PAF system in ultraviolet-B-induced epidermal cell interleukin-8 biosynthesis using a novel model system created by retroviral-mediated transduction of the PAF-receptor-negative human epidermal cell line KB with the human PAF receptor. Treatment of PAF-receptor-expressing KB cells with the metabolically stable PAF receptor agonist carbamoyl-PAF resulted in increased interleukin-8 mRNA and protein, indicating that activation of the epidermal PAF receptor was linked to interleukin-8 production. Ultraviolet B irradiation of PAF-receptor-expressing KB cells resulted in significant increases in both interleukin-8 mRNA and protein in comparison to ultraviolet-B-treated control KB cells. Pretreatment with PAF receptor antagonists inhibited both carbamoyl-PAF-induced and ultraviolet-B-induced interleukin-8 production in the PAF-receptor-positive cells, but not in control KB cells. Similarly, treatment of the PAF-receptor-expressing primary cultures of human keratinocytes or the human epidermal cell line A-431 with carbamoyl-PAF or ultraviolet B radiation resulted in interleukin-8 production that was partially inhibited by PAF receptor antagonists. These studies suggest that the epidermal PAF receptor may be a pharmacologic target for ultraviolet B radiation in skin and thus may act to augment ultraviolet-B-mediated production of cytokines such as interleukin-8.

Expression of progesterone receptor in human keratinocytes.

Despite the various responses of human skin to female sex hormones, cellular and subcellular targets and the mechanisms of action of estrogen and progesterone in human skin are not well understood. The detection of estrogen receptor (ER) and progesterone receptor (PR) in the skin is of great importance to understand the effect of estrogen and progesterone. In primary cultures of human keratinocytes, expression of ER and PR was monitored by immunocytochemistry and reverse transcriptase polymerase chain reaction (RT-PCR). Paraffin embedded skin tissues were stained with monoclonal antibodies to human ER and PR by immunohistochemistry. Cultured human keratinocytes expressed cytoplasmic PR protein and PR mRNA transcripts. By contrast, ER was detected only at the mRNA level. Suprabasal keratinocytes from samples of pruritic urticarial papules, plaques of pregnancy (PUPPP) and psoriasis were stained positively only for PR, while those from samples of erythema nodosum were negative for both ER and PR. Lesional epidermis of PUPPP showed positive PR immunoreactivity, while nonlesional epidermis did not. No other cells in the normal human skin were stained with ER and PR. The present study suggests that by expressing PR human keratinocytes act as targets for progesterone action.

Trichostatin A Up-Regulates Human Papillomavirus Type 11 Upstream Regulatory Region-E6 Promoter Activity in Undifferentiated Primary Human Keratinocytes

Human papillomavirus (HPV) gene expression in squamous epithelia is differentiation dependent in benign patient lesions and in organotypic raft cultures of primary human keratinocytes (PHKs). Using the lacZ reporter in raft cultures, we previously showed that this transcriptional regulation of the HPV type 11 (HPV-11) enhancer-promoter located in the upstream regulatory region (URR) appears to have resulted from coordination between the transcription transactivators AP1, Oct1, and Sp1 in differentiated upper strata and the repressor C/EBP in proliferating basal cells. We report here that trichostatin A, a specific inhibitor of histone deacetylase, dramatically stimulated reporter gene activity from the wild-type HPV-11 URR or the C/EBP mutation in PHKs grown in undifferentiated submerged cultures. In epithelial raft cultures, up-regulation occurred predominantly in basal and parabasal strata; this effect was promoter specific, as expression of the lacZ reporter gene driven by the murine leukemia virus long terminal repeat (LTR), the keratin 14 promoter, or the involucrin promoter was not altered, nor was expression of endogenous keratin 10 and profilaggrin affected. However, the responses were not cell type or species specific, as identical results were observed for both HPV-11 URR-lacZ and LTR-lacZ in murine retrovirus producer cell lines of fibroblast origin.

Production of TNF-alpha by primary cultures of human keratinocytes challenged with loxosceles gaucho venom

Primary cultures of human keratinocytes were challenged with increasing doses from 10 ng/mL to 2 &mgr;g/mL of Loxosceles gaucho venom, responsible for dermonecrotic lesion in humans. TNF-alpha was investigated by bioassay and ELISA in the supernatant of the cultures challenged with 100 ng/mL, 500 ng/mL, 1 and 2 &mgr;g/mL of venom. TNF-alpha was detected by bioassay in the supernatant of cultures challenged with 100 ng/mL, after 6 h. The cytokine was detected by ELISA in the supernatant of the cells challenged with doses of l &mgr;g/mL, after 6 and 12 h. The results point out the capacity of this venom to activate the keratinocytes in primary cultures to produce TNF-alpha. The production of cytokines could contribute to the local inflammatory process in patients bitten by Loxosceles sp.

A Distal Element in the HPV-11 Upstream Regulatory Region Contributes to Promoter Repression in Basal Keratinocytes in Squamous Epithelium

In benign squamous lesions and in organotypic epithelial cultures, the human papillomavirus (HPV) E6 and E7 genes are transcriptionally up-regulated in differentiated, spinous keratinocytes. We previously identified sequence elements in the enhancer-promoter regions of HPV types 18 and 11 important for this promoter regulation by using the bacterial LacZ reporter gene in stratified raft cultures of primary human keratinocytes (PHKs) or in submerged, proliferating cultures acutely transduced with recombinant retroviruses. Notably, mutations in the promoter-proximal Sp1, Oct1, and AP1 sites each significantly reduce reporter activity in differentiated cells, indicating that the bound factors are transcription transactivators. In the present study, we performed further mutagenesis on distal motifs in the HPV-11 regulatory region in PHKs in submerged and raft cultures. Mutations in an AP2-like site, three individual NF-1 sites, or five NF-1 sites collectively reduced promoter activity slightly in differentiated cells. A mutation in a putative glucocorticoid response element had no discernable effect in the presence or the absence of dexamethasone. However, mutations in a C/EBP binding site, especially the distal site, strikingly up-regulated reporter gene expression, particularly in basal and lower spinous cells, implicating bound protein as a transcription repressor. Collectively, these results demonstrate that the overall differentiation-dependent papillomaviral gene expression observedin vivoandin vitroinvolves promoter repression in the lower strata and activation in the upper, differentiated strata.

Alterations in cyclin-dependent kinase 2 function during differentiation of primary human keratinocytes.

Terminal differentiation of epithelial cells is intimately linked to cell-cycle withdrawal. The tight coupling of these two processes is critical to maintenance of epidermal tissue homeostasis and is frequently disrupted in squamous cell carcinoma. To identify possible molecular targets of epithelial carcinogenesis, we investigated the regulatory pathways that couple cellular differentiation and proliferation in primary cultures of human keratinocytes and found that the cyclin-dependent kinase inhibitors (CKIs) p21cip1/waf1 and p27kip1 were induced early during differentiation of human keratinocytes, whereas p15ink4B was induced later in differentiation. The induction of p21c1/waf1 was mediated by both transcriptional and non-transcriptional mechanisms, and the activities of cyclin A/cyclin-dependent kinase (cdk) 2 and cyclin E/cdk2 complexes were specifically inhibited during keratinocyte differentiation. In contrast, p21cip1/wafl did not associate with cdk4, and the activities of cdk4 complexes remained unchanged. Hence, our results support the model that multiple CKIs participate in linking cellular proliferation and differentiation in human keratinocytes by specific modulation of cdk2 activity.

Arsenic Enhancement of Skin Neoplasia by Chronic Stimulation of Growth Factors

Although numerous epidemiological studies have shown that inorganic arsenicals cause skin cancers and hyperkeratoses in humans, there are currently no established mechanisms for their action or animal models. Previous studies in our laboratory using primary human keratinocyte cultures demonstrated that micromolar concentrations of inorganic arsenite increased cell proliferation via the production of keratinocyte-derived growth factors. As recent reports demonstrate that overexpression of keratinocyte-derived growth factors, such as transforming growth factor (TGF)-alpha, promote the formation of skin tumors, we hypothesized that similar events may be responsible for those associated with arsenic skin diseases. Thus, the influence of arsenic in humans with arsenic skin disease and on mouse skin tumor development in transgenic mice was studied. After low-dose application of tetradecanoyl phorbol acetate (TPA), a marked increase in the number of skin papillomas occurred in Tg.AC mice, which carry the v-Ha-ras oncogene, that received arsenic in the drinking water as compared with control drinking water, whereas no papillomas developed in arsenic-treated transgenic mice that did not receive TPA or arsenic/TPA-treated wild-type FVB/N mice. Consistent with earlier in vitro findings, increases in granulocyte/macrophage colony-stimulating factor (GM-CSF) and TGF-alpha mRNA transcripts were found in the epidermis at clinically normal sites within 10 weeks after arsenic treatment. Immunohistochemical staining localized TGF-alpha overexpression to the hair follicles. Injection of neutralizing antibodies to GM-CSF after TPA application reduced the number of papillomas in Tg.AC mice. Analysis of gene expression in samples of skin lesions obtained from humans chronically exposed to arsenic via their drinking water also showed similar alterations in growth factor expression. Although confirmation will be required in nontransgenic mice, these results suggest that arsenic enhances development of skin neoplasias via the chronic stimulation of keratinocyte-derived growth factors and may be a rare example of a chemical carcinogen that acts as a co-promoter.

Post-transcriptional induction of p21cip1 protein by human papillomavirus E7 inhibits unscheduled DNA synthesis reactivated in differentiated keratinocytes.

Productive infection by human papillomaviruses (HPVs) occurs only in differentiated squamous epithelial cells in papillomas, condylomata, and low grade intraepithelial neoplasias. Host DNA replication is reactivated in a fraction of terminally differentiated keratinocytes in benign human lesions and in organotypic raft cultures of primary human keratinocytes (PHKs) transduced with retroviruses expressing HPV-18 E7 oncogene from its native upstream regulatory region (URR). Thus the natural function of E7 protein, which inactivates pRB family proteins, is to induce host genes essential to support viral DNA replication in post-mitotic cells. Using this raft culture model system, we show that HPV-18 URR-E7 induces the universal cyclin-dependent kinase inhibitor p21cip1 protein in a fraction of differentiated PHKs. Induction is mediated by posttranscriptional mechanisms independent of p53. Double immunofluorescence studies demonstrate that, in raft cultures and in laryngeal papillomas, p21cip1 induction and reactivated host DNA synthesis take place in a mutually exclusive manner in PCNA-positive, differentiated keratinocytes. We suggest that p21cip1 induction effectively blocks unscheduled DNA synthesis reactivated by E7. These results begin to explain the inverse relationship between p21cip1 induction and HPV activities previously observed in a spectrum of benign lesions regardless of HPV types present.

Biphasic Effect of Exogenous Nitric Oxide on Proliferation and Differentiation in Skin Derived Keratinocytes but Not Fibroblasts

Nitric oxide (NO) is known to exert cytotoxic and cytostatic effects in various cells and tissues. Although NO formation in human skin has been convincingly demonstrated, little is known about the NO-mediated effects in skin physiology and pathology. Here, we investigate the influence of NO on proliferation, differentiation, and apoptosis of primary cultures of normal human keratinocytes and fibroblasts. Four different NO donors at concentrations ranging from 0.01 to 5 mM were added every 12 h or 24 h to primary cultures of human keratinocytes and fibroblasts, and cells cultured for up to 3 d in the presence of these compounds. Cultures were examined for necrosis or apoptosis using trypan blue exclusion and in situ nick-translation. Cultures were also screened for the expression of the proliferation marker Ki67 and for an increase in cell numbers using neutral red staining. In addition, keratinocytes were stained for cytokeratin 6 expression to assess differentiation. We find that both keratinocytes and fibroblasts are highly resistant towards necrosis- or apoptosis-inducing effects of NO. In both cell types NO modulates cell growth, albeit in a cell-type specific pattern: cytostasis becomes significant in fibroblasts at concentrations of > or = 0.25 mM of the NO donor. In keratinocytes a biphasic effect is found with increased proliferation at low concentrations ranging from 0.01 to 0.25 mM and cytostasis at concentrations of > or = 0.5 mM. Conversely, expression of cytokeratin 6 is decreased at the lower NO donor concentrations and increased at higher concentrations as an indication of induction of differentiation at higher NO concentrations. Collectively, our results demonstrate that NO modulates proliferation and differentiation in human skin cells, a finding that will help to explain the pathophysiology of human skin diseases. Moreover, these findings suggest that NO generation in human skin diseases is not directly associated with local cell destruction, in contrast to findings in several other human diseases.

A proinflammatory activity of interleukin 8 in human skin: expression of the inducible nitric oxide synthase in psoriatic lesions and cultured keratinocytes.

Psoriasis is a common chronic skin disease mediated by cellular immune mechanisms and characterized by an intense neutrophil cell infiltrate and proliferative activation of epidermal keratinocytes. We have previously described the expression of the inducible nitric oxide synthase (iNOS) in epidermal keratinocytes of psoriatic skin lesions. In this study, the role of iNOS in psoriatic inflammation was explored ex vivo in psoriatic skin biopsies and in vitro in primary cultures of human keratinocytes. Messenger RNA for the iNOS enzyme (iNOS mRNA) was detected by reverse transcriptase polymerase chain reaction in skin biopsies from patients with psoriasis, but not in skin specimens from patients with atopic eczema or from healthy volunteers. As demonstrated by in situ hybridization and immunohistochemistry, expression of iNOS mRNA and its gene product was localized to the epidermal keratinocytes of psoriatic skin lesions. In situ hybridization further revealed a complete colocalization of mRNA expression for iNOS with interleukin (IL) 8 receptor-specific mRNA either in the basal germinative cell layer or at focal sites of ongoing neutrophil inflammation in suprabasal cell layers. Because psoriatic keratinocytes have previously been shown to express mRNA transcripts for IL-8, it seemed reasonable to hypothesize that iNOS expression could be induced in an autocrine loop by IL-8. This hypothesis was substantiated by our in vitro experiments showing that a combination of IL-8 and interferon gamma induces the expression of iNOS-specific mRNA and of the functional enzyme in cultured human keratinocytes. These results suggest an important role for iNOS in concert with IL-8 and its receptor early during the formation of psoriatic lesions.

Intracellular redistribution of Ku immunoreactivity in response to cell-cell contact and growth modulating components in the medium.

Ku is a heterodimeric protein first recognized as a human autoantigen but now known to be widely distributed in mammalian cells. Analysis of repair-deficient mutant cells has shown that Ku is required for DNA repair, and roles in DNA replication and transcription have also been suggested on the basis of in vitro observations. Ku is generally regarded as a nuclear component. However, in the present paper, we show that a quantitatively significant fraction (half or more) of Ku is located in the cytoplasm of cultured primate cells, and that major changes in epitope accessibility of both nuclear and cytoplasmic Ku components are associated with the transition from sparse to confluent cell densities. The same changes in immunoreactivity were seen in HeLa, 293, CV-1 (monkey) and HPV-transformed keratinocyte cell lines, and in primary cultures of human keratinocytes. The immunostaining pattern of sparsely grown cells could be converted to the 'confluent' configuration by re-plating them at the same low density on a monolayer of mouse 3T3 cells. The confluent antigen pattern could also be induced in sparse cells within 15-30 minutes by exposure of the cells to serum- or Ca(2+)-free medium or overnight with 2 mM hydroxyurea. Somatostatin at 0.12 mM blocked the effects of serum/Ca2+ deprivation of Ku p70 antigen distribution in sparse CV-1 cells, and in confluent cultures reversed the usual nuclear concentration of p70 immunoreactivity. However, somatostatin did not alter the expected immunostaining patterns of p86. Preliminary studies indicate that sparse CV-1 cells, but not HeLa cells, respond to as little as 1 pM of TGF-beta 1 in the culture medium by the rapid appearance of nuclear immunoreactivity. TGF-alpha had no apparent effect. These findings are consistent with the participation of Ku in a signal transduction system responsive to the inhibitory effect of cell-cell contact on the one hand and to cytokines and growth-supportive components of the culture medium on the other.