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 critical role in cell differentiation, and it has been shown to confer resistance to apoptosis. Based on this, and on evidence that activation of the gp130/signal transducer and activator of transcription (STAT) signal transduction pathway by interleukin (IL)-6 type cytokines promotes differentiation and prevents apoptosis in osteoblastic cells, we have investigated the possibility that p21 is a downstream effector of this 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-stimulated MG63 cells formed protein-DNA complexes with a 19-base pair nucleotide 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, overexpression of Stat3 potentiated the cytokine effect on the p21 promoter; whereas a dominant negative Stat3, or a mutation of the STAT response element on the promoter, significantly reduced the cytokine effect. Finally, antisense oligonucleotides complementary to p21 mRNA inhibited OSM-induced stimulation of alkaline phosphatase expression and antagonized the protective effect of OSM on anti-Fas-induced apoptosis. These results demonstrate that p21 is a downstream effector of gp130/Stat3 activation and a critical mediator of the pro-differentiating and anti-apoptotic effects of IL-6 type cytokines on human osteoblastic cells.
Bellido
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T
Division of Endocrinology and Metabolism, the Center for Osteoporosis and Metabolic Bone Diseases, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. tmbellido@life.uams.edu
O'Brien
C A
CA
Roberson
P K
PK
Manolagas
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SC
D015703:Antigens, CD / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology; D001483:Base Sequence; D002454:Cell Differentiation / Q000502:physiology; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics*; D050822:Cytokine Receptor gp130; D006801:Humans; D015850:Interleukin-6 / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000378:metabolism; D016376:Oligonucleotides, Antisense; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D016159:Tumor Suppressor Protein p53 / Q000502:physiology; D015854:Up-Regulation / Q000502:physiology*
D015703:Antigens, CD; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; C494199:IL6ST protein, human; D015850:Interleukin-6; D008562:Membrane Glycoproteins; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D050822:Cytokine Receptor gp130