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 transformation and early adenovirus gene transactivation functions of the ElA region were analyzed with deletion and point mutations. Deletion of amino acids from position 86 through 120 had little effect on the lytic or transforming functions of the ElA products, while deletion of amino acids from position 121 through 150 significantly impaired both functions. The sensitivity of the transformation function to alterations in the region from amino acid position 121 The ElA and E1B genes of adenovirus types 2 and 5 (Ad2 and Ad5) are sufficient for complete transformation of rodent cells (6,16,17,33,47,55). In addition, the ElA gene alone is capable of producing a partially transformed phenotype in rat cells (24,43). ElA products are also involved in the regulation of expression of other adenovirus gene products during productive infection in human cells. In the absence of functional ElA products, expression of other early genes and transcription from the major late promoter are reduced (1,26,32,37,57).Early in infection two mRNAs are made from ElA, the 12S and 13S products (2,7,28,39). These products are predicted to encode proteins 243 and 289 amino acids long, respectively, which differ only by the presence of the 46 amino acids at positions 140 through 185 which are unique to the 13S product (Fig. 1).The 13S product has been shown to be sufficient for the activation of other adenovirus genes required for lytic infection of HeLa cells (5,9,16,25,35,36,41,52,60,63). Several reports have indicated that both the 12S and 13S products have partial transformation activities. Both the 12S and 13S products have been shown to induce immortalization of rodent cells, although some differences have been noted in the phenotypes of the cells immortalized by the separate products of the ElA region (20,25,34,60,63). Our experiments and those of others indicate that the 12S product does not efficiently activate other adenovirus genes in HeLa cells (20,34,36,52,63). One interpretation of these results is that at least some aspects of the transformation functions reside in the region common to the 12S and 13S products, while the 13S unique region is required in addition for efficient activation of the early adenovirus genes. The importance of the 13S unique region for efficient activation of adenovirus early genes is further emphasized by the demonstration that single amino acid changes in the 13S unique region severely impair the ability of the ElA region to activate adenovirus early genes (13). Other evidence indicates that the 12S product actually does contain a significant ability to transactivate adenovirus early genes (11,31,60). No mutations have been obtained which specifically impair the immortalization function of the ElA products, so the question is still unresolved whether the transformation and early adenovirus gene transactivating functions are separable in the ElA products.To learn more about the relationship between the transformation and early adenovirus gene transactivation functions of the 12S and 13S ElA ...

We are using viral oncogene probes to study the pathways by which osteoblast-specific gene expression is induced in ascorbic acid-treated MC3T3-E1 cells. The 12S product of the adenovirus E1A gene binds directly to key cellular regulators and, as a result, represses tissue specific gene expression and blocks differentiation in a wide variety of cell types. The main cellular targets of the E1A 12S product are the pRB family and p300/CBP family. The p300 family appears to be the primary target for E1A-mediated repression of tissue-specific gene expression in a variety of cell types. We have generated MC3T3-E1 cell lines that stably express either the wild-type 12S product or a mutant that targets p300/CBP, but not the pRB family. Using these constructs to dissect osteoblast differentiation, we found that targeting of p300/CBP appears to be sufficient to repress alkaline phosphatase expression, although a low but functional level of expression can be maintained if the pRB family is not targeted as well. Induction of alkaline phosphatase expression and activity can be dissociated from expression of late-stage markers such as osteocalcin and osteopontin. Surprisingly, cell lines exhibiting severe repression of alkaline phosphatase activity differentiate to a mineral-secreting phenotype much like normal MC3T3-E1 cells. Osteopontin induction is dependent on at least a minimal level of alkaline phosphatase activity, although it is not dependent on induction of alkaline phosphatase at the RNA level. If alkaline phosphatase is supplied exogenously, osteopontin expression can be induced in conditions in which endogenous alkaline phosphatase is severely repressed.

Plasmids expressing partial adenovirus early region 1A (ElA) coding sequences were tested for activities which facilitate in vitro establishment (immortalization) of primary baby rat kidney cells and which enable the T24 Harvey ras-related oncogene and the polyomavirus middle T antigen (pmt) gene to transform primary baby rat kidney cells. ElA cDNAs expressing the 289-and 243-amino acid proteins expressed both ElA transforming functions. Mutant hrA, which encodes a 140-amino acid protein derived from the amino-terminal domain shared by the 289-and 243-amino acid proteins, enabled ras (but not pmt) to transform and facilitated in vitro establishment to a low, but detectable, extent. These studies suggest that ElA functions which collaborate with ras oncogenes and those which facilitate establishment are linked. Furthermore, ElA transforming functions are not associated with activities of the 289-amino acid ElA proteins required for efficient transcriptional activation of viral early region promoters.

We have analyzed the ceH cycle effects that different domains of the adenovirus EIA proteins have on quiescent primary BRK cells. Studies with deletion mutants that in combination removed all but the N-terminal 85 amino acids common to both the 12S and 13S proteins suggest that this region may be sufficient for the induction of synthesis of proliferating cell nuclear antigen and the stimulation of DNA synthesis. A second domain also common to the N-terminal exon of the 12S and 13S proteins was required for the induction of mitosis and stimulation of proliferation of primary BRK cells. A virus containing a mutation in this region was still able to stimulate DNA synthesis efficiently. A third domain, unique to the 13S protein, was required for the accelerated activation of the cellular thymidylate synthase gene in a manner similar to the 13S-dependent stimulation of adenovirus early region genes.

Adenovirus early region 1A (E1A) oncogene-encoded sequences essential for transformation- and cell growth-regulating activities are localized at the N terminus and in regions of highly conserved amino acid sequence designated conserved regions 1 and 2. These regions interact to form the binding sites for two classes of cellular proteins: those, such as the retinoblastoma gene product, whose association with the E1A products is specifically dependent on region 2, and another class which so far is known to include only a large cellular DNA-binding protein, p300, whose association with the E1A products is specifically dependent on the N-terminal region. Association between the E1A products and either class of cellular proteins can be disrupted by mutations in conserved region 1. While region 2 has been studied intensively, very little is known so far concerning the nature of the essential residues in the N-terminal region, or about the manner in which conserved region 1 participates in the binding of two distinct sets of cellular proteins. A combination of site-directed point mutagenesis and monoclonal antibody competition experiments reported here suggests that p300 binding is dependent on specific, conserved residues in the N terminus, including positively charged residues at positions 2 and 3 of the E1A proteins, and that p300 and pRB bind to distinct, nonoverlapping subregions within conserved region 1. The availability of precise point mutations disrupting p300 binding supports previous data linking p300 with cell cycle control and enhancer function.

Among the various biological activities expressed by the products of the adenovirus EIA gene are the abilities to induce cellular DNA synthesis and proliferation in quiescent primary baby rat kidney cells. The functional sites for these activities lie principally within two regions of the ElA proteins: an N-terminal region and a small second region of approximately 20 amino acids further downstream. To study the biological functions of the first domain, we constructed an in-frame deletion of amino acid positions 23 through 107 of the ElA products. This deletion did not impede the ability of the ElA products to transactivate the adenovirus early region 3 promoter in a transient-expression assay in HeLa cells. The ability to induce DNA synthesis in quiescent baby rat kidney cells was, however, lost in the absence of these sequences. Deletion of the small second region induced a form of S phase in which DNA synthesis occurred in the apparent absence of controls required for the cessation of DNA synthesis and progression through the remainder of the cell cycle. These cells did not appear to accumulate in or before G2, and many appeared to have a DNA content greater than that in G2. The functions of both domains are required for production of transformed foci in a ras cooperation assay. Focus formation occurred, however, even when the two domains were introduced on two separate plasmids. This complementation effect appeared to require expression of both of the mutant proteins and did not appear to result merely from recombination at the DNA level.

To distinguish the individual roles of the 13S, 12S, and 9S adenovirus ElA gene products, we isolated the corresponding cDNA clones and recombined them into both plasmids and viruses. Only the expected ElA mRNA products were made from the corresponding 12S and 13S viruses. The 9S mRNA was detected when the 9S virus was coinfected with the 13S virus but not when either virus was infected alone. The 13S virus formed plaques equally well in 293 cells, HeLa cells, and A549 cells, a human lung oat cell carcinoma line. Plaque titers of the 12S virus were much reduced in HeLa and A549 cells compared with 293 cells, although the 12S virus is multiplicity-dependent leaky in both HeLa and A549 cells. A549 cells were significantly more permissive than HeLa cells for growth of the 12S virus. In A549 cells even at low multiplicities of infection the final yield of 12S virus eventually approached the maximum yield from 293 cells. Expression from the adenovirus early region 2 and early region 3 promoters in HeLa cells was activated in the presence of a 13S cDNA ElA region but not in the presence of a 12S ElA cDNA region. Although defective for lytic growth in HeLa cells, the 12S virus immortalized BRK cells at very high efficiency, whereas infection of these cells with 13S virus, as with wild-type ElA virus, resulted mainly in cell death. The 13S product does have an immortalization function, however, revealed in the absence of adenovirus lytic functions when a plasmid containing the ElA 13S cDNA region was transfected into BRK cells. The 9S virus failed to immortalize infected BRK cells or to interfere with focus formation when coinfected with the 12S virus.