The clonal osteoblastic cell line MC3T3-El has been used as a model to study the mechanisms of interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF) action on type I collagen and prostaglandin H synthase transcription in bone cells. Using MC3T3-El cells permanently transfected with ColCAT 3.6, a chimeric gene consisting of 3.6 kb of alpha1(I) flanking sequence directing transcription of the structural thloramphenicol acetyltransferase (CAT) gene, we found that cytokines regulate alpha1(I) promoter activity in a differentiation-dependent manner. In cells plated at high density, which exhibit an osteoblastic phenotype and express high levels of CAT activity, IL-1 and TNF caused repression of alpha1(I) transcription. On the other hand, in cells plated at low density, which are less differentiated, a stimulatory transcriptional response to cytokines was observed. Phorbol 12-myristate 13-acetate (PMA), which mimics IL-1 and TNF actions in some systems, repressed ColCAT 3.6 activity regardless of the differentiation state. However, a dramatic stimulatory effect of PMA, but not cytokines, was unmasked in differentiated cells harboring a 5'-deletion construct which lacks the upstream basal enhancer region of the alpha1(I) promoter. The location of cytokine and PMA response elements in the alpha1(I) promoter will be mapped using a functional CAT assay. Initially, a series of 5' deletion mutants will be tested for cytokine and PMA responsiveness. ColCAT 3.6 mutants containing internal deletions will be prepared to assess the role of downstream sequences in the presence of the intact upstream enhancer. Putative response elements will be tested for their ability to confer responsiveness to a heterologous thymidine kinase promoter. Regions responsive to IL-1, TNF or PMA will be examined for constitutive or inducible DNA-protein interactions by mobility shift, DNase protection and methylation interference analysis. The function of putative response elements identified by these methods will be assessed by site-directed mutagenesis. The 5' flanking region of the PGH synthase gene, which encodes the rate-limiting enzyme in prostaglandin biosynthesis, will be cloned and sequenced. Regulation of PGH synthase transcription by cytokines will be analyzed in a manner similar to that described for the alpha1(I) promoter. These studies should contribute to understanding the mechanisms by which cytokines alter type I collagen and prostaglandin synthesis in bone. Thus, these studies could have important implications for the pathogenesis of osteoporosis and bone loss associated with chronic inflammatory disease.
