The epidermis is a stratified squamous epithelium composed primarily of keratinocytes that become postmitotic and undergo sequential changes in gene expression during terminal differentiation. The expression of the transcription factor CCAAT/enhancer binding protein ␤ (C/EBP␤) within mouse epidermis and primary keratinocytes has recently been described; however, the function of C/EBP␤ within the epidermal keratinocyte is unknown. We report here that transient transfection of mouse primary keratinocytes with a C/EBPresponsive promoter-reporter construct resulted in a sevenfold increase in luciferase activity when keratinocytes were switched to culture conditions that induce growth arrest and differentiation. Forced expression of C/EBP␤ in BALB/MK2 keratinocytes inhibited growth, induced morphological changes consistent with a more differentiated phenotype, and upregulated two early markers of differentiation, keratin 1 (K1) and keratin 10 (K10) but had a minimal effect on the expression of late-stage markers, loricrin and involucrin. Analysis of the epidermis of C/EBP␤-deficient mice revealed a mild epidermal hyperplasia and decreased expression of K1 and K10 but not of involucrin and loricrin. C/EBP␤-deficient primary keratinocytes were partially resistant to calcium-induced growth arrest. Analysis of terminally differentiated spontaneously detached keratinocytes or those induced to differentiate by suspension culture revealed that C/EBP␤-deficient keratinocytes displayed striking decreases in K1 and K10, while expression of later-stage markers was only minimally altered. Our results demonstrate that C/EBP␤ plays an important role in the early events of stratified squamous differentiation in keratinocytes involving growth arrest and K1 and K10 expression.

No abstract

Virus replication in higher vertebrates is restrained by IFNs that cause cells to transcribe genes encoding antiviral proteins, such as 2 -5 oligoadenylate synthetases. 2 -5 oligoadenylate synthetase is stimulated by dsRNA to produce 5 -phosphorylated, 2 -5 -linked oligoadenylates (2-5A), whose function is to activate RNase L. Although RNase L is required for a complete IFN antiviral response and mutations in the RNase L gene (RNASEL or HPC1) increase prostate cancer rates, it is unknown how 2-5A affects these biological endpoints through its receptor, RNase L. Presently, we show that 2-5A activation of RNase L produces a remarkable stimulation of transcription (>20-fold) for genes that suppress virus replication and prostate cancer. Unexpectedly, exposure of DU145 prostate cancer cells to physiologic levels of 2-5A (0.1 M) induced approximately twice as many RNA species as it downregulated. Among the 2-5A-induced genes are several IFN-stimulated genes, including IFN-inducible transcript 1͞P56, IFN-inducible transcript 2͞P54, IL-8, and IFN-stimulated gene 15. 2-5A also potently elevated RNA for macrophage inhibitory cytokine-1͞non-steroidal antiinflammatory drug-activated gene-1, a TGF-␤ superfamily member implicated as an apoptotic suppressor of prostate cancer. Transcriptional signaling to the macrophage inhibitory cytokine-1͞nonsteroidal antiinflammatory drug-activated gene-1 promoter by 2-5A was deficient in HeLa cells expressing a nucleasedead mutant of RNase L and was dependent on the mitogenactivated protein kinases c-Jun N-terminal kinase and extracellular signal-regulated kinase, both of which were activated in response to 2-5A treatments. Because 2-5A and RNase L participate in defenses against viral infections and prostate cancer, our findings have implications for basic cellular mechanisms that control major pathogenic processes.prostate ͉ macrophage inhibotory cytokine 1 ͉ HPC1 ͉ RNASEL

The secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 (1,25D) has been shown to regulate the growth and differentiation of human prostate cancer (PCa) cells, although the precise molecular mechanisms mediating these effects have not been defined. Previous studies in our laboratory demonstrated that the antiproliferative effects of 1,25D on PCa cells are mediated through the nuclear vitamin D receptor (VDR). In the present study, we performed gene profiling of LNCaP human PCa cells following 1,25D treatment and identified the antitumorigenic gene, prostate derived factor (PDF), as being highly induced by 1,25D. PDF is a member of the TGF-beta superfamily and has been implicated in a variety of functions directly related totumorigenicity including antiproliferative and pro-apoptotic effects. Gene expression studies using 1,25D analogs and a VDR antagonist demonstrate that 1,25D-mediated induction of PDF message and protein in PCa cells is dependent on VDR action. PDF is a transcriptional target of the tumor suppressor, p53. Here we show that the expression of PDF in nine PCa cell lines is dependent on functional p53. Additionally, transfection of p53-null ALVA-31 PCa cells with a p53 expression plasmid, and expression of dominant negative p53 in LNCaP PCa cells, show that the ability of VDR to induce PDF requires functional p53. Importantly, forced PDF expression in PC-3 cells results in decreased cell proliferation, soft agar cloning, and xenograft tumor size. These data demonstrate that PDF exerts antitumorigenic properties on PCa cells and its regulation by 1,25D may provide insights into the action of 1,25D in PCa.

NAG-1 (nonsteroidal anti-inflammatory drug-activated gene), a member of the transforming growth factor ␤ superfamily, is involved in cellular processes such as inflammation, apoptosis/survival, and tumorigenesis and is regulated by p53, Sp1, and Egr-1. In the current study,

CCAAT/enhancer-binding protein ␣ (C/EBP␣), a basic leucine zipper transcription factor, is involved in mitotic growth arrest and differentiation. Given that numerous proteins involved in cell cycle regulation are degraded via the ubiquitin-proteasome system, we examined whether the C/EBP␣ protein is degraded via a proteasomal mechanism. In cycloheximide-treated BALB/MK2 keratinocytes we found that C/EBP␣ is a short-lived protein with a half-life of ϳ1 h. Treatment with proteasome inhibitors, MG-132 or lactacystin, blocked the degradation of the C/EBP␣ protein. Higher molecular weight species of ubiquitinated C/EBP␣ were detected in BALB/MK2, and in vitro studies confirmed that C/EBP␣ is degraded by the proteasome in an ATPand ubiquitin-dependent manner. GSK3 is a known C/EBP␣ kinase and treatment of keratinocytes with LiCl, an inhibitor of GSK3 resulted in: (i) a 5-fold increase in C/EBP␣ protein levels, (ii) increased electrophoretic mobility of C/EBP␣, and (iii) no increase in C/EBP␣ mRNA levels suggesting that GSK3-mediated phosphorylation of C/EBP␣ may target it for proteasomal degradation. However, a mutant C/EBP␣ containing T to A mutations in the GSK3 phosphorylation sites (T222A and T226A) retained its response to LiCl, and additional pharmacological inhibitors of GSK3 did not alter C/EBP␣ levels indicating the effects of LiCl on C/EBP␣ are GSK3-independent. LiCl treatment of BALB/ MK2 cells inhibited C/EBP␣ degradation and produced a 6-fold increase in the half-life of C/EBP␣ protein. In vitro studies revealed that LiCl inhibited proteasome activity and the ensuing degradation of C/EBP␣. These results demonstrate C/EBP␣ is degraded via a ubiquitindependent proteasomal pathway, and LiCl stabilizes C/EBP␣ through a GSK3-independent pathway involving direct inhibition of proteasome activity.

Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality. Recent studies have stated that NOTCH signaling plays an important role in the development and progression of CRC. However, the role of Jagged-2 (JAG2), one of the NOTCH ligands, has not been delineated in colorectal tumorigenesis and drug resistance. In the present study, we have examined the impact of targeting JAG2 on CRC cells. Among all the members of NOTCH ligands, only the expression of JAG2 was found up-regulated in the intestinal tumors of ApcMin/+ mice as compared to the nearby normal mucosa. JAG2 expression was also observed in a panel of human CRC cell lines. Pharmacological inhibition or genetic knockdown of β-catenin in CRC cell lines suppressed JAG2 expression, suggesting Wnt/β-catenin regulation of JAG2 expression. In addition, deletion of Apc gene in the intestinal cells of Apc conditional knockout mice resulted in up-regulation of JAG2 expression. Modulation of JAG2 expression significantly affected in vivo tumorigenicity of CRC cell lines. Moreover, knockdown of JAG2 sensitized CRC cells to chemotherapeutic agents, while ectopic expression of JAG2 increased chemoresistance of the CRC cells. Significant down-regulation of p21 was observed in JAG2-knockdown cells. Forced expression of p21 rescued the sensitivity of JAG2-knockdown cells to doxorubicin. In addition, the chemosensitivity of p21-null cells was not affected by JAG2 knockdown. These results suggest that JAG2 modulates the sensitivity of CRC cells to chemotherapeutic agents through p21. Our study identifies JAG2 as a novel target for therapeutic intervention of CRC.

Background Nonsteroidal anti-inflammatory drug-activated gene (NAG-1), a divergent member of the transforming growth factor beta superfamily, has been implicated in many cellular processes, including inflammation, early bone formation, apoptosis, and tumorigenesis. Recent clinical studies suggests that a C to G single nucleotide polymorphism at position 6 (histidine to aspartic acid substitution, or H6D) of the NAG-1 protein is associated with lower human prostate cancer incidence. The objective of the current study is to investigate the activity of NAG-1 H6D variant in prostate cancer tumorigenesis in vivo. Methods Human prostate cancer DU145 cells expressing the H6D NAG-1 or wild-type NAG-1 were injected subcutaneously into nude mice and tumor growth was monitored. Serum and tumor samples were collected for subsequent analysis. Results The H6D variant was more potent than the wild-type NAG-1 and inhibited tumor growth significantly compared to control mice. Mice with tumors expressing the wild-type NAG-1 have greater reduced both body weight and abdominal fat than mice with H6D variant tumors suggesting different activities of the wild-type NAG-1 and the H6D NAG-1. A significant reduction in adiponectin, leptin and IGF-1 serum levels was observed in the tumor bearing mice with a more profound reduction observed with expression of H6D variant. Cyclin D1 expression was suppressed in the tumors with a dramatic reduction observed in the tumor expressing the H6D variant. Conclusion Our data suggest that the H6D variant of NAG-1 inhibits prostate tumorigenesis by suppressing IGF-1 and Cyclin D1 expression but likely additional mechanisms are operative.