TRANSCRIPTIONAL ACTIVATION OF THE P21(WAF1,CIP1,SDI1) GENE BY INTERLEUKIN-6 TYPE CYTOKINES - A PREREQUISITE FOR THEIR PRO-DIFFERENTIATING AND ANTI-APOPTOTIC EFFECTS ON HUMAN OSTEOBLASTIC CELLS

The cyclin-dependent kinase inhibitor p21(WAF1,CIP1,SDI1) plays a crit ical role in cell differentiation, and it has been shown to confer res istance to apoptosis. Based on this, and on evidence that activation o f the gp130/signal transducer and activator of transcription (STAT) si gnal transduction pathway by interleukin (IL)-6 type cytokines promote s differentiation and prevents apoptosis in osteoblastic cells, we hav e investigated the possibility that p21 is a downstream effector of th is signaling pathway in osteoblasts. We report that either oncostatin M (OSM) or IL-6 plus soluble IL-6 receptor increased the levels of p21 mRNA and protein in the osteoblast-like human osteosarcoma cell line MG63 and stimulated the activity of a 2.4-kilobase pair segment of the human p21 gene promoter. Further, nuclear extracts from cytokine-stim ulated MG63 cells formed protein-DNA complexes with a 19-base pair nuc leotide fragment of the p21 promoter containing a single STAT response element. The identity of the binding proteins as Stat3 and Stat1 was demonstrated with specific antibodies. In addition, and in support of a mediating role of STATs in the activation of the p21 promoter, overe xpression of Stat3 potentiated the cytokine effect on the p21 promoter ; whereas a dominant negative Stat3, or a mutation of the STAT respons e element on the promoter, significantly reduced the cytokine effect. Finally, antisense oligonucleotides complementary to p21 mRNA inhibite d OSM-induced stimulation of alkaline phosphatase expression and antag onized the protective effect of OSM on anti-Fas-induced apoptosis. The se results demonstrate that p21 is a downstream effector of gp130/Stat 3 activation and a critical mediator of the pro-differentiating and an ti-apoptotic effects of IL-6 type cytokines on human osteoblastic cell s.