Phosphate is a specific signal for induction of osteopontin gene expression

Abstract

Osteopontin is a phosphorylated glycoprotein secreted to the mineralizing extracellular matrix by osteoblasts during bone development. It is believed to facilitate the attachment of osteoblasts and osteoclasts to the extracellular matrix, allowing them to perform their respective functions during osteogenesis. Several other functions have been suggested for this protein, and its up-regulation is associated with various disease states related to calcification, including arterial plaque formation and the formation of kidney stones. Although expression of this gene has been demonstrated in multiple tissues, its regulation is not well understood. Our previous studies on the roles of the retinoblastoma protein (pRB) and p300͞CBP in the regulation of osteoblast differentiation revealed a link between osteopontin induction and the synthesis of alkaline phosphatase. In this paper, we describe results specifically linking induction of osteopontin to the enzymatic activity of alkaline phosphatase in the medium, which results in the generation of free phosphate. This elevation of free phosphate in the medium is sufficient to signal induction of osteopontin RNA and protein. The strong and specific induction of osteopontin in direct response to increased phosphate levels provides a mechanism to explain how expression of this product is normally regulated in bone and suggests how it may become up-regulated in damaged tissue. A s cells undergo terminal differentiation, various markers are induced in an ordered and sequential manner, but required steps in the induction process are not always clear because individual events in the sequence are not easily separable. The biology of the DNA tumor virus oncogene, adenovirus E1A, points to the cellular E1A targets, retinoblastoma protein (pRB) and p300͞CBP, as important regulators of gene expression during terminal differentiation (1-4). We have taken advantage of E1A genetics to explore the roles of the pRB and p300͞CBP protein families in expression of early and late markers during osteoblast differentiation. The MC3T3-E1 cell line is derived from newborn mice calvaria (5). It is an established cell line, but the cells maintain much of the tightly linked controls between proliferation and differentiation that usually are seen only in primary cells (6). Treatment with ascorbic acid stimulates these cells to differentiate along the osteoblast line (6-8). Induced cells deposit a collagenous extracellular matrix, accompanied by the activation of specific genes associated with the osteoblast phenotype, such as alkaline phosphatase, osteocalcin, and osteopontin. If a source of organic phosphate such as ␤-glycerol phosphate is present, a discrete zone of hydroxyapatitecontaining mineral is formed within the collagen fibrils. The sequence from induction to mineralization proceeds in a tightly regulated order over a span of 2 to 3 weeks (see schematic in Fig. 1), which permits a detailed analysis of the order of events.We previously have characterized two MC3T3-E1 cell lines stably express...

The Mechanism of Phosphorus as a Cardiovascular Risk Factor in CKD

“…10,11,22 These studies 11,22 have established that the action of phosphorus is related to the activity of the Pit1 sodium-dependent transporter in VSMC, as suggested in osteoblasts. 48 Its role as a signal molecule may be through Pit1-induced activation of a membrane-associated signaling complex similar to that known to associate with the sodium-dependent transport protein of the proximal tubule and osteoclasts, NaPi2a. 49 Our data in vivo were directly related to our in vitro model.…”

Differentiation of MC3T3-E1 on poly(4-styrenesulfonic acid-co-maleic acid)sodium salt-coated films

“…OPN is a phosphorylated glycoprotein which contains a string of polyaspartic acid residues as well as a arginineglycine-aspartate (RGD) sequence. OPN is believed to facilitate the attachment of osteoblasts and osteoclasts to the extracellular matrix, allowing them to perform their respective functions during osteogenesis 35) . BSP is a highly glycosylated and sulphated phosphoprotein that is almost exclusively found in mineralized connective tissues such as bone and cementum.…”

Current Understanding of the Pathogenesis of Progressive Chronic Kidney Disease in Cats

“…18,184,185 Extracellular phosphate concentrations have also been associated with increased production of profibrotic mediators and may have a direct stimulatory effect on RAAS. [186][187][188] There is some evidence supporting the role of phosphorus in the pathogenesis of feline CKD. Early studies evaluating phosphate restricted diets showed that cats on restricted diets had reduced evidence of renal histopathological lesions (calcification, fibrosis and inflammatory cell infiltration) compared to cats on a high phosphorus diet.…”