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

Abstract

Mineralization, a characteristic phenotypic property of osteoblastic lineage cells, was blocked by 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) and decanoyl-Arg-Arg-Leu-Leu-chloromethyl ketone (dec-RRLLcmk), inhibitors of SKI-1 (site 1; subtilisin kexin like-1) protease. Because SKI-1 is required for activation of SREBP and CREB (cAMP-response element-binding protein)/ATF family transcription factors, we tested the effect of these inhibitors on gene expression. AEBSF decreased expression of 140 genes by 1.5-3.0-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, whereas 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, overexpression 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.Bone formation and mineralization is a multistep process of differentiated osteoblastic cells. Based upon an analysis of gene expression during osteogenesis, Lian and Stein (1) noted that "peak levels of expressed genes reflect a developmental sequence of bone cell differentiation characterized by three principal periods: proliferation, extracellular matrix maturation, and mineralization, and two restriction points to which the cells can progress but cannot pass without further signals." These authors defined these three periods of osteoblastic differentiation in terms of patterns of sequential gene expression. For example, fibronectin and type I collagen expression denoted the pattern of pre-osteoblastic cells, alkaline phosphatase, and matrix GLA protein reflected the matrix maturation stage, and osteopontin and osteocalcin reflected the mineralization stage of mature osteoblastic cells (1). However, the number of genes required for normal bone formation and mineralization has grown rapidly to include Phex (Phex), dentin matrix protein1 (Dmp1), and type XI collagen (COL11).Phex (phosphate-regulating endopeptidase homolog, Xlinked) is a transmembrane metalloendoprotease enriched on bone osteoblasts and osteocytes (2). It is essential for phosphate homeo...

Deletion of Mbtps1 (Pcsk8, S1p, Ski-1) Gene in Osteocytes Stimulates Soleus Muscle Regeneration and Increased Size and Contractile Force with Age

“…Interestingly, MBTPS1 is required for the transcription of a number of bone matrix and mineralization-related genes including type XI collagen, Phex, Dmp1, fibronectin, and fibrillin in bone osteoblasts and osteocytes. Correspondingly, inactivation of MBTPS1 by AEBSF or specific inhibitor decanoyl-Arg-Arg-Leu-Leu-chloromethyl ketone blocks transcription of the aforementioned genes and inhibits mineralization (13,14). These results demonstrated that the differentiated phenotype of osteoblastic cells and possibly osteocytes depends upon MBTPS1.…”

“…Although microCT analyses revealed no differences in trabecular or cortical bone mineral density or morphological parameters between cKO and control tibiae, three-point bending studies on femora demonstrated a significant 25% gain in stiffness in Mbtps1-deficient bone (p ϭ 0.01). We rationalize this outcome in terms of a requirement for MBTPS1 for transcription of bone matrix genes including fibronectin, fibrillin2, and collagen XI (13). Because expression was not down-regulated in osteocyte-enriched bone (array data deposited at NCBI under accession number GSE69975), MBTPS1 regulation of these mineralization-related genes may be restricted to osteoblast-like cells (13).…”

“…52 g of bone extracts were electrophoresed per lane of 4 -20% linear gradient SDS gels (Precise Tris-HEPES gels, Pierce) at 50 V for 2.5 h and then electroblotted onto PVDF membrane at 100 V for 2 h. Coomassie Blue prestained globular protein standards were also electrophoresed on each gel. Blots were processed for immunodetection with rabbit anti-human or mouse MBTPS1 C-terminal primary antibodies (21) using a SuperSignal West Femto Maximum Sensitivity Chemiluminescent Substrate kit (Thermo Scientific) as described previously (13). Blots were digitally imaged using a Fuji LAS4000 system with a charge-coupled device camera.…”

“…Protein content of extracts was determined with the Non-Interfering Protein Analysis Assay kit. Proteins were solubilized in SDS-and 8 M urea-containing sample buffer and electrophoresed under reducing conditions on 4 -20% gradient gels (Precise, Thermo Fisher) as described previously (13). Gels were then electroblotted onto PVDF membranes in a CAPS/MeOH buffer, pH 11.0 and subjected to immunodetection with a 1:20,000 dilution of primary antibody recognizing the C-terminal domain of MBTPS1 (21).…”

“…Therefore, it is apparent that the 18-kDa fragment is enriched in bone osteocytes, whereas the 65-kDa form is present in muscle. Although MBTPS1 is a membrane-bound protease localized to the cis/medial Golgi, a soluble form can also be shed into the medium of kidney cells (30) and bone cells (13).…”