Mutant envelope residues confer a transactivation function onto N-terminal sequences of the v-Rel oncoprotein.

The retroviral oncoprotein v-Rel is a member of the Rel/ NF-kappaB family of transcription factors. v-Rel has multiple changes as compared to the proto-oncoprotein c-Rel, and these changes render v-Rel highly oncogenic in avian lymphoid cells. Previous results have shown that three mutant residues in the eleven helper virus-derived Envelope (Env) amino acids (aa) at the N-terminus of v-Rel are required for its full oncogenicity. In this report, we show that these mutant Env aa also enable sequences in the N-terminal half of v-Rel to activate transcription in yeast and chicken cells, under conditions where the analogous sequences from c-Rel either do not or only weakly activate transcription. Removal of the Env aa from v-Rel or site-directed mutations that revert the three mutant residues to the residues present in the Rev-A helper virus Env protein abolish this transactivation ability of v-Rel. Addition of mutant Env aa onto c-Rel is not sufficient to fully restore the transactivation function; other sequences in the N-terminal half of v-Rel are needed for full transactivating ability. A C terminally-truncated form of NF-kappaB p100 (p85), produced in HUT-78 human leukemic cells, also activates transcription in yeast, under conditions where the normal p52 and p100 proteins do not. Furthermore, transcriptional activation by p85 in yeast is likely to occur through N-terminal sequences. Taken together, these results are consistent with a model in which transactivation by N-terminal Rel Homology (RH) domain sequences in oncogenic Rel family proteins is influenced by sequences outside the RH domain.

A role for interleukin-1 in epidermal differentiation: regulation by expression of functional versus decoy receptors.

Although human epidermis contains levels of interleukin-1 (IL-1) up to 100 times higher than other tissues, the role of this cytokine in epidermal biology is unknown. Here, we show that interleukin-1 regulates the expression of mRNAs for two proteins associated with the differentiated phenotype of human keratinocytes, cellular retinoic acid-binding protein type II (CRABP II) and small, proline rich protein 1 (SPRR1). The ability of IL-1 to induce these transcripts correlates directly with keratinocyte expression of the IL-1 receptor type I (IL-1 RI) during differentiation and inversely with the expression of the type II IL-1 receptor (IL-1 RII), shown in other cell types to be a nonfunctional, decoy receptor. Furthermore, addition to keratinocyte cultures of an IL-1 RI-blocking, but not an IL-1 RII-blocking, antibody reduces the levels of CRABP II and SPRR1 mRNAs in these cells. These data suggest that epidermal IL-1 functions to promote keratinocyte differentiation and that a change in the IL-1 receptor profile of these cells initiates this IL-1 response through a relative enhanced expression of functional IL-1 receptors.

In vivo and in vitro SPRR1 gene expression in normal and malignant keratinocytes.

The small proline-rich protein 1 (SPRR1) gene encodes a precursor of the keratinocyte cornified envelope. To understand SPRR1 regulation we investigated its expression and modulation in keratinocytes in vivo and in vitro. SPRR1 was strongly expressed in suprabasal layers of the epidermis in newborn skin but only weakly expressed in adult skin. Both in vivo and in vitro, SPRR1 was not expressed in undifferentiated cells of basal or squamous carcinomas. However, within the same tumors and in premalignant lesions of squamous cell origin, cells with histologic evidence of differentiation showed a relative increase in SPRR1 transcript level. Within 24 h physiologic doses of uv irradiation induced SPRR1 mRNA in vivo. To investigate the possibility that SPRR1 expression is regulated by uv-induced cytokines, keratinocytes were stimulated with interleukin-1 (IL-1) and interleukin-3 (IL-3). Both significantly induced SPRR1 mRNA, while TGF-beta, known to lower IL-1 receptor in keratinocytes, down-regulated it. Moreover, proximity to inflammatory cells in vivo was associated with SPRR1 induction in anaplastic tumor cells. Our data suggest that SPRR1 is induced early in differentiation of normal keratinocytes but is not expressed in anaplastic cells of keratinocyte origin. Further, its regulation in skin appears to be modulated at least in part through cytokine release.

Epidermal differentiation enhances CRABP II expression in human skin.

The cellular retinoic acid-binding proteins (CRABP I and II) are thought to mediate the effects of retinoic acid on target cells. We have used riboprobes complementary to CRABP I and II mRNAs to study the expression of these messages in normal and abnormal human skin. CRABP II was expressed predominantly in the suprabasal layers of the epidermis, with stronger expression in newborn than in sun-protected adult skin. Interestingly, the epidermis adjacent to or overlying squamous cell or basal cell carcinomas also showed strong expression, whereas the tumor cells were negative, with the exception of more differentiated cells surrounding the "keratin pearls" within squamous cell carcinomas. CRABP II mRNA was also found in the more differentiated cells of the hair follicles, in the outer root sheath. CRABP I message was undetectable in the epidermis or in the dermis of normal skin but was detected in the cells of the papillary dermis surrounding basal and squamous cell carcinomas. These data suggest that increased levels of CRABP II mRNA accompany keratinocyte differentiation in vivo.

Gene modulation accompanying differentiation of normal versus malignant keratinocytes.

Using tetradecanoylphorbol 13-acetate (TPA), a known inducer of epithelial cell differentiation, and Northern blot analysis, we investigated in normal versus well-differentiated malignant keratinocytes the modulation of genes implicated in their growth or differentiated function. In normal keratinocytes transient c-fos induction was detected within 30 min after stimulation and was followed by rapid down regulation of the proto-oncogenes c-myc and epidermal growth factor receptor. Within hours after stimulation mRNA levels for three well-characterized differentiated keratinocyte products, involucrin, interleukin-1 beta (IL1-beta), and fibronectin, were induced, as were those for a growth arrest and DNA damage-inducible (GADD 153) gene and a small proline-rich (SPR1) gene, both known to be associated with differentiation but with of as yet unknown functions. Heat shock protein 70 gene was initially down regulated and was induced only after 48 h. The well-differentiated malignant keratinocyte cell line differed in that the c-fos, GADD, SPR1, and IL1-beta genes had several-fold higher induction, but involucrin mRNA was undetectable and fibronectin mRNA was only minimally induced after TPA stimulation. Malignant cells reached terminal differentiation faster than normal keratinocytes as measured by inability to exclude trypan blue dye, and in situ hybridization using a riboprobe for the differentiation-associated SPR1 gene showed that normal keratinocytes constitutively express this transcript while malignant keratinocytes with virtually identical morphology and growth rate do not. These studies greatly expand our understanding of gene activation and down regulation during normal keratinocyte differentiation and imply that a malignant cell line, even when retaining the phenotype of normal cells, differs in its response to outside stimuli, furnishing at best an imperfect model for investigating the molecular mechanisms of cellular differentiation.