Osteoblast autonomous Pi regulation via Pit1 plays a role in bone mineralization.

The complex pathogenesis of mineralization defects seen in inherited and/or acquired hypophosphatemic disorders suggests that local inorganic phosphate (P(i)) regulation by osteoblasts may be a rate-limiting step in physiological bone mineralization. To test whether an osteoblast autonomous phosphate regulatory system regulates mineralization, we manipulated well-established in vivo and in vitro models to study mineralization stages separately from cellular proliferation/differentiation stages of osteogenesis. Foscarnet, an inhibitor of NaP(i) transport, blocked mineralization of osteoid formation in osteoblast cultures and local mineralization after injection over the calvariae of newborn rats. Mineralization was also down- and upregulated, respectively, with under- and overexpression of the type III NaP(i) transporter Pit1 in osteoblast cultures. Among molecules expressed in osteoblasts and known to be related to P(i) handling, stanniocalcin 1 was identified as an early response gene after foscarnet treatment; it was also regulated by extracellular P(i), and itself increased Pit1 accumulation in both osteoblast cultures and in vivo. These results provide new insights into the functional role of osteoblast autonomous P(i) handling in normal bone mineralization and the abnormalities seen in skeletal tissue in hypophosphatemic disorders.

Individual osteoblasts in the developing calvaria express different gene repertoires.

Several studies in vitro and a few in vivo have suggested that mature osteoblasts heterogeneously express osteoblast markers. In one recent study of the osteoblasts associated with bone nodules formed in vitro in rat calvaria cell populations, extensive diversity was documented in the overall gene repertoires expressed. To address whether comparable heterogeneity is evident in vivo, we investigated the expression of nine osteoblast lineage markers by both in situ hybridization and immunohistochemistry. At 21 days of fetal rat development, the calvaria is a rapidly growing bone with distinct maturational zones that are readily observed in coronal sections; that is, an osteogenic front emerging at sagittal and coronal sutures is adjacent to areas of growing trabeculae of bone, followed by more mature areas of remodeling bone. Based on expression patterns, markers can be divided into two categories. One category comprises markers that are globally expressed by all osteoblasts irrespective of their position in the calvaria. Of those tested, only two, alkaline phosphatase and the pth/pthrp receptor, fit into this category. All other markers analyzed, including transcription factors (c-fos and msx-2), matrix molecules (bone sialoprotein, osteopontin, and osteocalcin), and a hormone (pthrp), were differentially expressed only in subpopulations of osteoblasts, based on cell maturational status, environment (ectocranial vs. endocranial surfaces), and microenvironment (adjacent osteoblasts). Preosteoblasts and osteocytes in different regions of the calvaria also expressed different subsets of the lineage markers. Mechanisms responsible for generating differential gene expression profiles appear to be both transcriptional and posttranscriptional. These results indicate that postproliferative, morphologically indistinguishable osteoblasts are not a homogeneous class of cells, but instead are molecularly diverse. The present results also raise the possibility that lineage progression and/or maintenance of the differentiated state may be adaptable in the calvaria.

cDNA fingerprinting of osteoprogenitor cells to isolate differentiation stage-specific genes.

A cDNA fingerprinting strategy was developed to identify genes based on their differential expression pattern during osteoblast development. Preliminary biological and molecular staging of cDNA pools prepared by global amplification PCR allowed discrim-inating choices to be made in selection of expressed sequence tags (ESTs) to be isolated. Sequencing of selected ESTs confirmed that both known and novel genes can be isolated from any developmental stage of interest, e.g. from primitive progenitors, intermediate precursors or mature osteoblasts. EST expression provides insight into possible interrelated physiological functions and putative interacting molecules during differentiation. This method offers a functional genomics approach to isolate differentiation stage-specific genes in samples as small as a single cell.

Polymerase chain reaction-based technique for the selective enrichment and analysis of mosaic arg201 mutations in G alpha s from patients with fibrous dysplasia of bone.

Mutations in the arg201 codon of the alpha s G protein-subunit have been associated with a variety of disorders, but analysis of such mutations has been complicated by their mosaic presentation. To overcome the problems associated with the analysis of genomic mutations that may be present in low and variable yield throughout the body, a polymerase chain reaction (PCR)-based technique has been developed that allows the selective amplification of products from the mutant allele. This technique uses site-directed mutagenesis to generate a PCR product from the normal allele that is susceptible to restriction endonuclease digestion, whereas that from the mutant allele is resistant to digestion. Consecutive and repeated cycles of amplification and digestion allow selective enrichment of the product from the mutant allele. The technique has been applied to the analysis of patients with fibrous dysplasia of bone, where the consequence of G alpha s mutations may vary from monostotic to polyostotic lesions, and has been performed with DNA isolated from either bone biopsy specimens or peripheral blood leukocytes. In addition to the previously described arg-->his and arg-->cys substitutions, the analyses have detected a novel arg-->ser substitution in one of the patients. This patient presented with a panostotic disease and may represent a unique subgroup of fibrous dysplasia.

New mechanisms of regulation of the genomic actions of vitamin D in bone cells: interaction of the vitamin D receptor with non-classical response elements and with the multifunctional protein, calreticulin.

Vitamin D exerts its genomic effects following binding to a specific receptor which is a member of the steroid hormone receptor superfamily. The vitamin D receptor (VDR) forms heterodimers with retinoid X receptors (RXRs) and the dimer then interacts with its cognate binding site, termed vitamin D response element (VDRE), to affect the transcription of target genes. Recent studies have identified novel sequence motifs for VDREs as well as novel protein-protein interactions involving the VDR. These will be reviewed with particular emphasis on the complex VDRE from the c-fos proto-oncogene promoter region and the inhibition of the vitamin D signal transduction pathway by the multifunctional protein, calreticulin. Thus research on the control of gene transcription by vitamin D reveals examples of molecular interplay between transcriptional regulatory pathways and provides new insight into the molecular mechanism of action of vitamin D.

A composite element binding the vitamin D receptor, retinoid X receptor alpha, and a member of the CTF/NF-1 family of transcription factors mediates the vitamin D responsiveness of the c-fos promoter.

The hormonal form of vitamin D, 1 alpha,25-dihydroxyvitamin D3 [1,25- (OH)2D3], transiently stimulates the transcription of the c-fos proto-oncogene in osteoblastic cells. We have identified and characterized a vitamin D response element (VDRE) in the promoter of c-fos. The 1,25-(OH)2D3-responsive region was delineated between residues -178 and -144 upstream of the c-fos transcription start site. A mutation that inhibited binding to the sequence concomitantly abolished 1,25-(OH)2D3-induced transcriptional responsiveness; similarly, cloning to the site upstream of a heterologous promoter conferred copy-number-dependent vitamin D responsiveness to a reporter gene, demonstrating that we have identified a functional response element. The structure of the c-fos VDRE was found to be unusual. Mutational analysis revealed that the c-fos VDRE does not conform to the direct repeat configuration in which hexameric core-binding sites are spaced by a few nucleotide residues. In contrast, the entire 36-bp sequence was essential for binding. We identified the vitamin D receptor and the retinoid X receptor alpha as components of the complex that bound the c-fos VDRE. However, our results also show that a putative CCAAT-binding transcription factor/nuclear factor 1 (CTF/NF-1) family member bound the response element in conjunction with the nuclear hormone receptors. The expression of this CTF/NF-1 family member appeared restricted to bone cells. These data hint at new molecular mechanisms of action for vitamin D.

Increased expression of the c-fos proto-oncogene in bone from patients with fibrous dysplasia.

BACKGROUND: Fibrous dysplasia is characterized by intense marrow fibrosis and increased rates of bone turnover. The lesions of fibrous dysplasia resemble those described in the long bones of transgenic mice overexpressing the c-fos proto-oncogene. Activating mutations in the alpha subunit of the stimulatory guanine-nucleotide-binding protein (GS alpha) linked to adenylate cyclase have recently been described in bone cells from patients with the McCune-Albright syndrome and fibrous dysplasia. METHODS: We used in situ hybridization to determine the level of expression of c-fos in bone-biopsy specimens from two normal subjects, eight patients with fibrous dysplasia, and six patients with other bone disorders characterized by high rates of bone turnover. The probe used corresponded to the fourth exon of the c-fos gene. RESULTS: High levels of c-fos expression were detected in the bone lesions from all eight patients with fibrous dysplasia. No expression of c-fos was detected in bone specimens from the normal subjects or from specimens of normal bone obtained from patients with fibrous dysplasia. The cells that expressed c-fos in the dysplastic lesions were fibroblastic and populated the marrow space. A very low level of c-fos expression was detected in the biopsy specimens from the patients with other bone diseases. One patient with polyostotic fibrous dysplasia and one patient with the McCune-Albright syndrome were tested for the previously described GS alpha gene mutations and were found to express these mutations in bone. CONCLUSIONS: Increased expression of the c-fos proto-oncogene, presumably a consequence of increased adenylate cyclase activity, may be important in the pathogenesis of the bone lesions in patients with fibrous dysplasia.

Differential stimulation of fos and jun family members by calcitriol in osteoblastic cells.

We have looked at the effects of calcitriol (1 alpha,25-dihydroxyvitamin D3) on the expression of the members of the fos and jun families of protooncogenes in an osteoblastic cell line and in primary cultures of osteoblasts. Calcitriol treatment of starved, confluent cultures of MC3T3-E1 cells induced a rapid and transient stimulation of the expression of c-fos, fos-B, c-jun, and jun-B with varying kinetics. The expression of fra-1 and jun-D was not affected by calcitriol in those cells. The selective stimulation of fos and jun family members by calcitriol was also observed in primary cultures of osteoblasts isolated from newborn mouse calvaria, suggesting that this modulation is a physiological response of the bone cells and not an artefact of the established cell line. The calcitriol effect was specific and dose-dependent. The expression of the c-Fos protein correlated with the expression of the mRNA in calcitriol-treated cells. The calcitriol-induced stimulation of c-fos expression was modulated, at least in part, at the level of the initiation and elongation of transcription, whereas its effects on c-jun and jun-B expression was controlled at the posttranscriptional level by a mechanism that does not implicate stabilization of their respective mRNAs. The differential stimulation of the expression of certain members of the fos and jun families by calcitriol support a role for these oncoproteins in bone cell physiology.