Inhibition of Proprotein Convertase Ski-1 Blocks Transcription of Key Extracellular Matrix Genes Regulating Osteoblastic Mineralization

Mineralization, a characteristic phenotypic property of osteoblastic lineage cells, was blocked by AEBSF and dec-RRLL-cmk, inhibitors of SKI-1 (site 1; subtilisin kexin like-1) protease. Since SKI-1 is required for activation of SREBP and CREB/ATF family transcription factors, we tested the effect of these inhibitors on gene expression. AEBSF decreased expression of 140 genes by 1.5- to 3.0-fold including Phex , Dmp1 , COL1A1 , COL11A1 and fibronectin . Direct comparison of AEBSF and dec-RRLL-cmk, a more specific SKI-1 inhibitor, demonstrated that expression of Phex , Dmp1 , COL11A1 and fibronectin was reduced by both while COL1A2 and HMGCS1 were reduced only by AEBSF. AEBSF and dec-RRLL-cmk decreased the nuclear content of SKI-1 activated forms of transcription factors SREBP-1, SREBP-2, and OASIS. In contrast to AEBSF, the actions of dec-RRLL-cmk represent the sum of its direct actions on SKI-1 and indirect actions on caspase-3. Specifically, dec-RRLL-cmk reduced intracellular caspase-3 activity by blocking the formation of activated 19 kDa caspase-3. Conversely, over-expression of SKI-1 activated SREBP-1a and CREB-H in UMR106-01 osteoblastic cells increased the number of mineralized foci and altered their morphology to yield mineralization nodules, respectively. In summary, SKI-1 regulates the activation of transmembrane transcription factor precursors required for expression of key genes required for mineralization of osteoblastic cultures in vitro and bone formation in vivo . Our results indicate that the differentiated phenotype of osteoblastic cells, and possibly osteocytes, depends upon the non-apoptotic actions of SKI-1.