The proto-oncogene c-fos is a major nuclear target for signal transduction pathways involved in the regulation of cell growth, differentiation, and transformation. Using the multistep skin carcinogenesis model, we have directly tested the ability of c-fos-deficient mice to develop cancer. Upon treatment with a tumor promoter, c-fos knockout mice carrying a v-H-ras transgene were able to develop benign tumors with similar kinetics and relative incidence as wild-type animals. However, c-fos-deficient papillomas quickly became very dry and hyperkeratinized, taking on an elongated, horny appearance. While wild-type papillomas eventually progressed into malignant tumors, c-fos-deficient tumors failed to undergo malignant conversion. Experiments in which v-H-ras-expressing keratinocytes were grafted onto nude mice suggest that c-fos-deficient cells have an intrinsic defect that hinders tumorigenesis. These results demonstrate that a member of the AP-1 family of transcription factors is required for the development of a malignant tumor.
The major di erentiation products of maturing keratinocytes contain AP-1 regulatory motifs, and AP-1 DNA binding activity increases in cultured keratinocytes induced to di erentiate by calcium. Here, we have analysed AP-1 transcriptional activity in mouse keratinocytes treated with calcium and 12-O-tetradecanoyl phorbol-13-acetate (TPA), two agents that induce terminal di erentiation of keratinocytes with di erent phenotypic consequences. Reporter constructs representing multimers of AP-1 sequences found in keratinocyte marker genes demonstrated that the calcium-induced AP-1 DNA binding activity does not correlate with transcriptional activation. Moreover, expression from active subunits of the pro®laggrin and spr 1 promoters increased in calcium-treated keratinocytes when the AP-1 sites were disrupted, indicating that AP-1 may negatively regulate certain promoters in these cells. In contrast, AP-1 reporter activity was increased in keratinocytes treated with TPA. This induction was dependent upon the expression of c-Fos since AP-1 transcriptional activity was not increased in TPA-treated keratinocytes derived from c-fos null mice. Analysis of AP-1 protein expression in calcium-and TPA-treated keratinocytes demonstrated that only TPA increased the expression of c-Jun, while Jun B and Jun D were induced by both of these agents. c-Fos was expressed only in TPA treated keratinocytes, Fra-2 was expressed only in calcium-treated cells, and Fra-1 was expressed in both. Exogenous expression of Fra-2 repressed AP-1 transcriptional activity in TPA-treated keratinocytes, while c-Fos expression activated the AP-1 sequence in calcium-treated keratinocytes. These data indicate that Fra-2 and c-Fos play opposing roles in regulating AP-1 activity in keratinocytes and that multiple inducerdependent regulatory pathways may exist for the expression of keratinocyte di erentiation markers.
Previously, we have shown that nuclear extracts from cultured mouse keratinocytes induced to di erentiate by increasing the levels of extra-cellular calcium contain Fra-1, Fra-2, Jun B, Jun D and c-Jun proteins that bind to the AP-1 DNA binding sequence. Despite this DNA binding activity, AP-1 reporter activity was suppressed in these cells. Here, we have detected the CREB family proteins CREB and CREMa as additional participants in the AP-1 DNA binding complex in di erentiating keratinocytes. AP-1 and CRE DNA binding activity correlated with the induction of CREB, CREMa and ATF-1 and CREB phosphorylation at ser 133 (ser 133 phospho-CREB) in the transition from basal to di erentiating keratinocytes, but the activity of a CRE reporter remained unchanged. In contrast, the CRE reporter was activated in the presence of the dominantnegative (DN) CREB mutants, KCREB and A-CREB, proteins that dimerize with CREB family members and block their ability to bind to DNA. The increase in CRE reporter activity in the presence of these mutants suggests that CRE-mediated transcriptional activity is suppressed in keratinocytes through protein-protein interactions involving a factor that dimerizes with the CREB leucine zipper. In experiments where the A-CREB mutant was co-transfected with an AP-1 reporter construct, transcriptional activity was also increased indicating that a CREB family member binds AP-1 sites and represses AP-1 transcriptional activity as well. Exogenous expression of the transcriptional repressor CREMa down-regulated both CRE and AP-1 reporters in keratinocytes suggesting that this factor may contribute to the suppression of AP-1 transcriptional activity observed in di erentiating keratinocytes.
Androgen exposure stimulates the growth of beard hair follicles. The follicle dermal papilla appears to be the site of androgen action; however, the molecular mechanisms that regulate this process are not well understood. In an attempt to identify genes that contribute to the androgen-responsive phenotype, we compared gene expression patterns in unstimulated and androgen-treated cultured human dermal papilla cells isolated from beard (androgen-sensitive) and occipital scalp (androgen-insensitive) hair follicles. Through this analysis, we identified three genes that are expressed at significantly higher levels in beard dermal papilla cells. One of these genes, sfrp-2 has been identified as a dermal papilla signature gene in mouse pelage follicles. Two of these genes, mn1 and atp1beta1, have not been studied in the hair follicle. A fourth, fibulin-1d, was slightly upregulated in beard dermal papilla cells. The differences in the expression of these genes in cultured beard and scalp dermal papilla cells reflected similar differences in microdissected dermal papilla isolated from intact beard and scalp follicles. Our findings introduce potentially novel signaling pathways in dermal papilla cells. In addition, this study supports that cultured dermal papilla cells provide a cell-based model system that is reflective of the biology of in vivo hair follicle cells.
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