FGF upregulates osteopontin in epiphyseal growth plate chondrocytes: Implications for endochondral ossification

Fibroblast growth factor receptor 3 (FGFR3) signaling pathways are essential for normal longitudinal bone growth. Mutations in this receptor lead to various human growth disorders, including Achondroplasia, disproportionately short-limbed dwarfism, characterized by narrowing of the hypertrophic region of the epiphyseal growth plates. Here we find that FGF9, a preferred ligand for FGFR3 rapidly induces the upregulation and secretion of the matrix resident phosphoprotein, osteopontin (OPN) in cultured chicken chondrocytes. This effect was observed as early as two hours post stimulation and at FGF9 concentrations as low as 1.25 ng/ml at both mRNA and protein levels. OPN expression is known to be associated with chondrocyte and osteoblast differentiation and osteoclast activation. Unexpectedly, FGF9 induced OPN was accompanied by inhibition of differentiation and increased proliferation of the treated chondrocytes. Moreover, FGF9 stimulated OPN expression irrespective of the differentiation stage of the cells or culture conditions. In situ hybridization analysis of epiphyseal growth plates from chicken or mice homozygous for the Achondroplasia, G369C/mFGFR3 mutation demonstrated co-localization of OPN expression and osteoclast activity, as evidenced by tartarate resistant acid phosphatase positive cells in the osteochondral junction. We propose that FGF signaling directly activates OPN expression independent of chondrocytes differentiation. This may enhance the recruitment and activation of osteoclasts, and increase in cartilage resorption and remodeling in the chondro-osseus border.     

Osteopontin is expressed by adult human osteoarthritic chondrocytes: protein and mRNA analysis of normal and osteoarthritic cartilage.

Osteopontin, a sulfated phosphoprotein with cell binding and matrix binding properties, is expressed in a variety of tissues. In the embryonic growth plate, osteopontin expression was found in bone-forming cells and in hypertrophic chondrocytes. In this study, the expression of osteopontin was analyzed in normal and osteoarthritic human knee cartilage. Immunohistochemistry, using a monoclonal anti-osteopontin antibody was negative on normal cartilage. These results were confirmed in Western blot experiments, using partially purified extracts of normal knee cartilage. No osteopontin gene expression was observed in chondrocytes of adult healthy cartilage, however, in the subchondral bone plate, expression of osteopontin mRNA was detected in the osteoblasts. In cartilage from patients with osteoarthritis, osteopontin could be detected by immunohistochemistry, Western blot analysis, in situ hybridization, and Northern blot analysis. A qualitative analysis indicated that osteopontin protein deposition and mRNA expression increase with the severity of the osteoarthritic lesions and the disintegration of the cartilaginous matrix. Osteopontin expression in the cartilage was limited to the chondrocytes of the upper deep zone, showing cellular and territorial deposition. The strongest osteopontin detection was found in deep zone chondrocytes and in clusters of proliferating chondrocytes from samples with severe osteoarthritic lesions. These data show the expression of osteopontin in adult human osteoarthritic chondrocytes, suggesting that chondrocyte differentiation and the expression of differentiation markers in osteoarthritic cartilage resembles that of epiphyseal growth plate chondrocytes.     

Okadaic acid stimulates osteopontin expression through de novo induction of AP-1

Osteopontin, a major non-collagenous bone matrix protein, is strikingly upregulated in various tissues under certain pathologic conditions, including cancer. However, the mechanism of upregulation of the osteopontin gene in tumor cells remains unclear. Okadaic acid, a strong non-phorbol ester tumor promoter, is known to stimulate the expression of osteopontin. The aim of the present study was to understand the mechanism by which okadaic acid regulates osteopontin gene expression. Okadaic acid stimulated osteopontin mRNA expression in several cell lines within 3 h, and the increase in osteopontin mRNA was sustained for 24 h. New protein synthesis was required for the okadaic acid-elicited increase in osteopontin mRNA expression. A serial promoter deletion study showed that the okadaic acid-response element is located between positions -265 and -73, a sequence that includes the Runx2, Ets-1, and AP-1 binding sequences. Okadaic acid increased the mRNA expression of AP-1 components but not of Runx2 or Ets-1. Site-directed mutagenesis and electrophoretic mobility shift assays confirmed that protein binding of the AP-1 consensus sequence is necessary for the okadaic acid-mediated osteopontin gene upregulation. These results indicate that de novo induction of the oncoprotein AP-1 is required for okadaic acid-stimulated osteopontin gene upregulation.     

In situ hybridization of bone matrix proteins in undecalcified adult rat bone sections.

We have developed a method for in situ hybridization of adult bone tissue utilizing undecalcified sections and have used it to histologically examine the mRNA expression of non-collagenous bone matrix proteins such as osteocalcin (bone Gla protein, BGP), matrix Gla protein (MGP), and osteopontin in adult rats. Expression was compared with that in bone tissues of newborn rats. In the adult bone tissue, osteocalcin mRNA was strongly expressed in periosteal and endosteal cuboidal osteoblasts but not in primary spongiosa near the growth plate. Osteopontin mRNA was strongly expressed in cells present on the bone resorption surface, osteocytes, and hypertrophic chondrocytes, but not in cuboidal osteoblasts on the formation surface. Osteopontin and osteocalcin mRNAs were expressed independently and the distribution of cells expressing osteopontin mRNA corresponded with acid phosphatase-positive mononuclear cells and osteoclasts. Expression of MGP mRNA was noted only in hypertrophic chondrocytes. In newborn rat bone tissues, expression of osteocalcin mRNA was much weaker than in adult rat bone tissues. These results clearly indicate the differential expression of mRNAs of non-collagenous bone matrix proteins in adult rat bone tissues.     

Structural characteristics of osteopontin mRNA]

Conservatism of primary and secondary structure of osteopontin mRNA in vertebrates of various taxes was studied using computer analysis and blot-hybridization procedure. Relatively high rate of evolution of a nucleotide sequence of osteopontin gene was revealed. Formation of hairpin-loop structures by adjacent sequences and a high content of AU-pairs indicate more flexible structure of the mammalian osteopontin mRNA as compared with the avian one. It has been detected some AU-enriched regions in 3'-terminal untranslated sequences, which presence is characteristic for short-lived mRNA. Evolutionary conserved subsequences in 3'- and 5'-terminal regions were suggested to be determinants of RNA-protein interaction in cytoplasm.     

Homocysteine attenuates the expression of osteocalcin but enhances osteopontin in MC3T3-E1 preosteoblastic cells

It has been pointed out that very high plasma levels of homocysteine are characteristic of homocystinuria, a rare autosomal recessive disease accompanied by the early onset of generalized osteoporosis. However, it is unclear by which mechanism hyperhomocysteine induces osteoporosis, although it is known to interfere with the formation of cross-links in collagen, an essential process in bone formation. Therefore, we investigated the effect of homcysteine on expression of osteocalcin and osteopontin in MC3T3-E1 preosteoblastic cells. Confluent cells were grown in RPMI 1640 containing 10% fetal calf serum with or without homocysteine in an atmosphere of 95% humidified air, 5% CO2 at 37 degrees C. The secretion of osteocalcin from the cells increased time-dependently until the end of culture (day 34), but 500 microM homocysteine led to an approximately 61% decrease for osteocalcin after 19 days of culture as compared with the control. On the other hand, osteopontin was not inhibited by 500 microM homocysteine but rather activated, and ranged from 134%-209% of the control level in the period from 10 days until the end of culture. From analysis of RT-PCR for mRNA of osteocalcin and osteopontin at the end of the culture, homocysteine levels of 100 and 500 microM significantly increased the expression of osteopontin mRNA with the control (p < 0.05). In contrast, the expression of osteopontin mRNA was suppressed in a dose-dependent manner, showing a mirror image of the effect on osteopontin mRNA. These findings suggest that hyperhomocystenemia appears to be an independent risk factor for osteoporosis by disturbing osteoblast function.     

Expression of transforming growth factor-beta s 1-4 in chicken embryo chondrocytes and myocytes

cDNA probes and antibodies for TGF-beta s 1, 2, 3, and 4 were used to study the expression of these different TGF-beta isoforms in cultured chicken embryo chondrocytes and cardiac myocytes, as well as in developing cartilage and heart tissues. TGF-beta s 2, 3, and 4 mRNAs, but not TGF-beta 1 mRNA, were detected in cultured chondrocytes and myocytes. Expression of TGF-beta s 2 and 4 mRNAs increased with age, while expression of TGF-beta 3 mRNA was independent of age in chondrocytes cultured from 12- to 17-day-old embryos. In contrast, expression of TGF-beta s 2, 3, and 4 mRNAs was constitutive in myocytes cultured from 7- to 9-day-old embryonic hearts; expression of TGF-beta s 3 and 4 mRNAs increased, while expression of TGF-beta 2 mRNA remained unchanged in myocytes from 10-day-old embryos. Immunoprecipitation studies demonstrated expression of TGF-beta in both the conditioned media and the cell lysates of metabolically labeled chondrocyte and myocyte cell cultures. Immunohistochemical staining of cultured chondrocytes and myocytes and of cartilage and heart tissues of developing chicken embryos with antibodies specific for each TGF-beta isoform showed immunoreactive TGF-beta s 1, 2, 3, and 4. Our results demonstrate coordinate expression of these four TGF-beta isoforms in chicken embryo chondrocytes and myocytes, both in vitro and in vivo, with expression of TGF-beta s 2, 3, and 4 mRNA and protein more prominent than that of TGF-beta 1.     

Expression of mRNA of murine bone-related proteins in ectopic bone induced by murine bone morphogenetic protein-4

To determine whether a system of ectopic bone formation induced by osteosarcoma-derived bone-inducing substance (bone morphogenetic protein-4) can be used as a model of developing bone at the molecular level, we studied the expression of bone-related protein mRNAs in the process of ectopic bone formation using non-radioisotopic in situ hybridization. Osteonectin mRNA was detected in fibroblast-like cells, which are similar to periosteal cells from the early to middle stages of bone development. The proportion of osteonectin mRNA-expressing cells was greater than that of osteopontin mRNA-expressing cells in hypertrophic chondrocytes and osteoblast-like cells. In contrast, osteopontin mRNA was localized in a limited population of hypertrophic chondrocytes, a single layer of osteoblast-like cells adjacent to the bone trabeculae in the middle stage of bone formation, and in a limited subset of osteocytes in the late stage. A strong osteocalcin mRNA signal was detected in osteoblast-like cells from the middle to late stages and in a limited subset of osteocytes in the late stage of bone development. Since the sequential gene expression pattern of bone-related proteins in the present system is comparable to that in embryonic osteogenesis, this system may be useful as a model for studying gene expression in osteogenesis.     

The effects of three different demineralization agents on osteopontin localization in adult rat bone using immunohistochemistry

Immunohistochemical localization of osteopontin, a phosphorylated acidic glycoprotein, was compared in adult rat femur fixed in 4% paraformaldehyde at 4° C for 48 h and demineralized at 4° C in ethylenediaminetetraacetic acid (EDTA), modified Jenkin's solution, or 15% formic acid, until radiographs indicated demineralization was complete. Formic acid was also evaluated at room temperature. EDTA solution (15 days) resulted in intense staining of osteocytes, periosteal osteoclasts and osteoblastic cells in osteonal bone. Osteoblasts were negative in the periosteum. No megakaryocyte staining was present; however, occasional neutrophils in the bone marrow were non-specifically stained. Demineralization in modified Jenkin's solution (16 days) showed osteopontin localization in bone matrix, hypertrophic and articular chondrocytes, and osteocytes. In cortical bone, almost all cement lines demarcating osteons showed very dense labeling. In the bone marrow, occasional megakaryocytes were immunopositive and neutrophils were non-specifically stained. Jenkin's produced non-specific staining of skeletal muscle and connective tissue. Formic acid demineralization (14 days, 4° C) resulted in osteopontin expression in osteoblasts, osteocytes, osteoclast precursors, bone matrix, osteoid, cement lines, and chondrocytes; osteoclasts, although present in very low numbers, were also positive. More labeled osteoblasts could be identified compared to Jenkin's demineralization. Also more intense non-specific staining of the bone marrow neutrophils was obtained than with Jenkin's. Harsh, rapid demineralization with formic acid (4 days, room temperature) produced a loss in antigenicity demonstrated by a reduction in staining intensity not experienced with the 4° C protocol; however, osteopontin was still localized in bone matrix and hypertrophic zone chondrocytes. These results indicate that demineralization is compatible with retention of immunoreactive osteopontin in adult rat bone. Both EDTA and formic acid demineralization produce excellent immunostaining and are preferred over the modified Jenkin's solution to minimize background levels of non-specific staining.     

Tissue specificity and developmental expression of rat osteopontin

Osteopontin is a 44 kd phosphoprotein abundant in bone matrix. We isolated a partial length cDNA for rat osteopontin and used it to examine its tissue specificity, its expression during bone development and its hormonal regulation. Osteopontin mRNA is most abundant in bone but is also found in considerable amounts in kidney. Osteopontin mRNA is regulated by the osteotropic hormones dexamethasone and 1,25(OH)2D3. Estimates of osteopontin mRNA levels indicate that the osteopontin gene is turned on relatively late in calvarial development.     

Fibroblast growth factor-2 regulates expression of osteopontin in periodontal ligament cells

Osteopontin is a protein found in the bone-related matrix and plays multiple regulatory roles in mineralizing and non-mineralizing tissue. In osteogenic cell-lines, the expression of osteopontin increases with the progression of differentiation, but both the expression and function of osteopontin vary with the cell type and its activation state. In this study, we examined the expression of osteopontin by clones established from mouse periodontal ligament, in response to inorganic phosphate and fibroblast growth factor (FGF)-2, which can induce periodontal tissue regeneration. The involvement of inorganic phosphate in the expression of osteopontin during the course of cell differentiation of a clone MPDL22 was confirmed by addition of foscarnet, an inorganic phosphate transport inhibitor. Although FGF-2 decreased the mRNA expression of almost every bone-related protein in MPDL22, FGF-2 upregulated the expression of osteopontin in MPDL22 at both mRNA and protein levels. Interestingly, FGF-2 enhanced the concentration of osteopontin in the culture supernatant of MPDL22, whereas inorganic phosphate did not. The FGF-2-induced osteopontin in the culture supernatant seems to be involved in cell survival activity. An immunohistochemical study showed that the FGF-2-induced osteopontin was mainly present in perinuclear matrices while the inorganic phosphate-induced osteopontin was associated with extracellular matrices in addition to perinuclear matrices. The present results indicated that FGF-2 induces unique expression of osteopontin, which may play a role different from the other bone-related proteins during the process of periodontal tissue regeneration by FGF-2.     

cDNA cloning, mRNA distribution and heterogeneity, chromosomal location, and RFLP analysis of human osteopontin (OPN)

A human osteopontin (OP) cDNA was isolated from a library made from primary cultures of human bone cells. The distribution of osteopontin mRNA in human tissues was investigated by Northern analysis and showed that the human message was predominant in cultures of bone cells and in decidua cells isolated at 6-12 weeks of gestation. Immunohistochemical analysis confirmed that OP expression is high in decidua cells as well as in the endometrial glands of a non-pregnant secretory-phase human uterus. Two variants of the OP message were evident on the basis of DNA sequencing and polymerase chain reaction amplification of bone and decidua cell mRNA. The peptides potentially translated by the variant messages differ by the presence (OP1b) or absence (OP1a) of 14 amino acids at residue 58 of the molecule. The deduced human protein sequence shows a conservation between species in the position of the Arg-Gly-Asp (RGD) cell attachment site. Chromosomal mapping of the osteopontin gene (OPN) using human-rodent cell hybrids demonstrated a location on chromosome 4 in the human genome. In situ hybridization of metaphase chromosomes using radiolabeled OP1a as a probe indicated that the gene is located on a region of 4q that is near the centromere. A high-frequency restriction fragment length polymorphism was evident in the DNA from 29 unrelated individuals using the enzyme BglII. Analysis of total genomic DNA by digestion with several restriction enzymes, Southern blotting, and hybridization with the human osteopontin cDNA indicated that the gene is a single copy with an approximate length of 5.4-8.2 kb.     

Chondrocyte protein with a poly-proline region is a novel protein expressed by chondrocytes in vitro and in vivo

Chondrogenic differentiation is a multistep process entailing the sequential activation and inhibition of the expression of a number of genes. To identify genes preferentially expressed at the hypertrophic stage rather than early differentiation stages of chicken chondrocyte differentiation, a subtracted cDNA library was generated. Here we describe the characterization of a cDNA isolated from this library and that of the encoded protein referred to as Chondrocyte Protein with a Poly-proline Region (CHPPR).The cDNA coding for CHPPR hybridizes with a 3.0-kb mRNA expressed at extremely low levels in dedifferentiated chondrocytes, cultured in adherent conditions, at low levels in differentiating chondrocytes and at very high levels in hypertrophic chondrocytes in suspension culture. The Parathyroid Hormone peptide [PTH (1-34)] enhances accumulation of CHPPR mRNA in cultured chondrocytes. This 3.0-kb mRNA is also detectable in several chick embryo tissues but at a lower extent when compared to that present in cartilage and in hypertrophic chondrocytes. The CHPPR cDNA has a complete open reading frame coding for a polypeptide with a calculated mass of 35.6 kDa containing a proline-rich region with a PPLP motif (single-letter amino acid code). We demonstrate by Western blot analysis that two CHPPR isoforms are detected in the cell lysates from cultured chondrocytes when they are not in the culture medium; furthermore, we find that the CHPPR gene is expressed in vivo by chick embryo chondrocytes at higher levels in the prehypertrophic and hypertrophic zones.     

Regulation of chicken ccn2 gene by interaction between RNA cis-element and putative trans-factor during differentiation of chondrocytes

CCN2/CTGF is a multifunctional growth factor. Our previous studies have revealed that CCN2 plays important roles in both growth and differentiation of chondrocytes and that the 3'-untranslated region (3'-UTR) of ccn2 mRNA contains a cis-repressive element of gene expression. In the present study, we found that the stability of chicken ccn2 mRNA is regulated in a differentiation stage-dependent manner in chondrocytes. We also found that stimulation by bone morphogenetic protein 2, platelet-derived growth factor, and CCN2 stabilized ccn2 mRNA in proliferating chondrocytes but that it destabilized the mRNA in prehypertrophic-hypertrophic chondrocytes. The results of a reporter gene assay revealed that the minimal repressive cis-element of the 3'-UTR of chicken ccn2 mRNA was located within the area between 100 and 150 bases from the polyadenylation tail. Moreover, the stability of ccn2 mRNA was correlated with the interaction between this cis-element and a putative 40-kDa trans-factor in nuclei and cytoplasm. In fact, the binding between them was prominent in proliferating chondrocytes and attenuated in (pre)hypertrophic chondrocytes. Stimulation by the growth factors repressed the binding in proliferating chondrocytes; however, it enhanced it in (pre)hypertrophic chondrocytes. Therefore, gene expression of ccn2 mRNA during endochondral ossification is properly regulated, at least in part, by changing the stability of the mRNA, which arises from the interaction between the RNA cis-element and putative trans-factor.     

Changes in osteopontin mRNA expression during phenotypic transition of rabbit arterial smooth muscle cells

 Transition from a contractile to a synthetic phenotype appears to be an early key event during the development of intimal thickening after arterial wall injury. We examined the expression of osteopontin mRNA, proliferation, and phenotypic properties of smooth muscle cells (SMCs) in rabbit neointima after balloon denudation and in primary culture. A strong osteopontin mRNA signal was detected in the thickened intima 1 week after balloon denudation and in the surface layer of the intima 2 weeks after balloon denudation. Ki-67 immunohistochemistry showed that osteopontin mRNA expression increased when SMCs entered the proliferating phase in the intima. Rabbit arterial SMCs on type I collagen after 1 day of primary culture with growth factors, as well as freshly isolated cells, were in the G₀ phase (contractile phenotype) and did not express osteopontin mRNA. After 3 days of culture, most cells entered the G_1B phase (synthetic phenotype) and expressed osteopontin mRNA. In the absence of growth factors, most cells transferred to the G_1A phase (intermediate phenotype) after 3 and 7 days, but did not express osteopontin mRNA. Our findings indicate that the osteopontin gene provides a marker that can be used to distinguish the phenotypic properties of vascular SMCs. Accepted: 22 November 1996