Acidic fibroblast growth factor signaling inhibits peroxynitrite-induced death of osteoblasts and osteoblast precursors.

After trauma injury to the musculoskeletal system, conditions such as ischemia and inflammation involve excess production of superoxide (O2*), nitric oxide (*NO), and their reaction product, peroxynitrite (ONOO-). Exposure of murine osteoblasts and rat-derived primary osteoblast precursors to ONOO- resulted in a dose- and time-dependent delayed cell death that was more characteristic of apoptosis than necrosis. Exposure of both cell populations to ONOO- immediately enhanced phosphorylation and nitration of tyrosine residues within several polypeptides. Treatment of osteoblasts and osteoblast precursors with exogenous acidic fibroblast growth factor (FGF-1) enhanced cellular growth, increased endogenous levels of tyrosine phosphorylation, and significantly induced expression of both osteopontin and osteocalcin messenger RNA (mRNA) as well as osteopontin protein. Pretreatment of both cell populations with exogenous FGF-1 prevented ONOO(-)-mediated death. Cell signaling induced by FGF-1 pretreatment had no major effect of total levels of tyrosine nitration after ONOO- treatment. Collectively, these in vitro efforts show that FGF-1 signaling renders osteoblasts and osteoblast precursors resistant to the cytotoxic effects of ONOO-. Consequently, results presented here predict the therapeutic use of this growth factor for promoting the progression of bone repair mechanisms after fracture trauma.

Validation of peripheral dual-energy X-ray absorptiometry for the measurement of bone mineral in intact and excised long bones of rats.

We evaluated the precision and accuracy of peripheral dual-energy X-ray absorptiometry (DXA) for the measurement of bone mineral density (BMD) and bone mineral content (BMC) in intact and excised femurs and tibias from rats. Thirty-one Sprague-Dawley rats (18F/13M; 114-360 g) were used in the study. Precision and accuracy were determined in 23 rats and prediction equations were evaluated in an independent sample of 8 animals. Precision was determined by measuring the right hindquarter three times with repositioning between scans. The femur and tibia were then excised, cleaned, and scanned in triplicate, with repositioning. CVs ranged from 0.66 to 2.24%. Accuracy of BMC was determined by comparison to bone ash values. BMC values for the intact and excised femur significantly overestimated bone ash (p < 0.001) by 33% and 5.5%, respectively. BMC for the intact tibia overestimated ash by 37% (p < 0.001), whereas BMC for the excised tibia underestimated ash by 1% (p < 0.05). However, BMC and bone ash were highly related for both bones, whether BMC was measured in the intact animal or after excision (r2 > 0.99). Cross-validation of prediction equations in an independent sample showed that there were no significant differences between predicted ash (based on BMC from DXA) and measured bone ash. These results suggest the peripheral DXA is a useful tool for measuring intact and excised rat leg bones.

Expression of osteopontin messenger RNA and protein in rheumatoid arthritis: effects of osteopontin on the release of collagenase 1 from articular chondrocytes and synovial fibroblasts.

OBJECTIVE: Osteopontin (OPN) is an extracellular matrix protein that has been implicated in the interactions between tumor cells and host matrix, including those involved in invasion and spread of tumor cells. Because joint destruction in rheumatoid arthritis (RA) is mediated by the invasive growth of synovial tissue through its attachment to cartilage, we examined the expression of OPN in the synovia of patients with RA and the effect of OPN on the production of collagenase 1 in rheumatoid synovial fibroblasts and articular chondrocytes. METHODS: The expression of OPN messenger RNA (mRNA) and protein in synovia from 10 RA patients was examined by in situ hybridization and immunohistochemistry. Synovial fibroblasts from RA patients and articular chondrocytes from patients without joint disease were cultured in the presence of various concentrations of OPN, and levels of collagenase 1 in the culture supernatants were measured by enzyme-linked immunosorbent assay. RESULTS: The expression of OPN mRNA and protein was observed in 9 of 10 specimens obtained from patients with RA. OPN was expressed in the synovial lining and sublining layer and at the interface of cartilage and invading synovium. Double labeling revealed that the majority of OPN-expressing cells were positive for the fibroblast-specific enzyme prolyl 4-hydroxylase and negative for the macrophage marker CD68, while only a few, single OPN-expressing cells were positive for CD68 at sites of synovial invasion into cartilage. OPN staining was not observed in lymphocytic infiltrates or leukocyte common antigen (CD45)-positive cells. Three of 3 cultures of human articular chondrocytes secreted detectable basal amounts of collagenase, with a dose-dependent increase upon OPN stimulation, while synovial fibroblast cultures produced much lower levels of collagenase, with only 2 of 4 fibroblast cultures responding in a dose-dependent manner. CONCLUSION: These findings suggest that OPN produced by synovial fibroblasts in the synovial lining layer and at sites of cartilage invasion not only mediates attachment of these cells to cartilage, but also contributes to matrix degradation in RA by stimulating the secretion of collagenase 1 in articular chondrocytes.

Bone response to titanium alloy implants placed in diabetic rats.

Although dental implants continue to provide consistent and predictable treatment options for most patients, some people with uncontrolled systemic disease may be denied implant treatment. Diabetes is one such disease. According to the U.S. Centers for Disease Control and Prevention, diabetes is a leading cause of blindness, kidney failure, and amputations of the lower extremities. These complications result from microvascular disturbances associated with diabetes. The effect of diabetes on the healing of titanium implants has not been well established. In this study of 32 rats, diabetes was induced in 16 animals by injection of streptozotocin (65 mg/kg); the remaining 16 animals served as controls. Titanium alloy implants were placed in the tibiae of all 32 rats using standard surgical techniques. Implants healed for 14 days. Blood samples were obtained for serum glucose, osteocalcin, and alkaline phosphatase analyses. Implants were retrieved and processed for histomorphometric analyses. Three quantities were measured using light microscopy, video capture, and computer analysis: percent osseointegration (i.e., linear bone interface), associated bone volume percent, and contact frequency. Diabetic animals demonstrated significantly less osseointegration than controls. However, bone volume percent in diabetic animals was about 4 times greater than controls. Biochemical analyses were mixed; diabetic animals demonstrated increased serum osteocalcin levels compared to controls but decreased alkaline phosphatase. Based on the results of this study, it was concluded that the bone response associated with titanium alloy implants in the tibiae of diabetic rats is uniquely different from controls.

Mechanisms of homocysteine toxicity on connective tissues: implications for the morbidity of aging.

It is proposed that chronic moderate hyperhomocysteinemia has a causal role in a number of common diseases of late life, including occlusive vascular disease, cognitive decline, senile osteoporosis and presbyopia. These diseases are seen as clinical counterparts of the main manifestations of homocystinuria (vascular occlusions of arteries and veins, mental retardation, osteoporosis and ectopia lentis, respectively) that develop only after many years of exposure to moderately elevated homocysteine (Hcy) levels. The multisystem toxicity of Hcy is attributed to its spontaneous chemical reaction with many biologically important molecules, primarily proteins. The formation of these Hcy-adducts is dependent on time and Hcy concentration and leads to loss or diminution of function of the derivatized molecules. Irreversible homocysteinylation of long-lived proteins should lead to cumulative damage and progressive clinical manifestations. Fibrillin 1 is seen as the paradigm of extracellular connective tissue proteins that are specially susceptible to Hcy (and presumably Hcy thiolactone) attack. The prominent presence of epidermal growth factor (EGF)-like domains in fibrillin and in many other extracellular proteins of the coagulation, anticoagulation, and lipoprotein transport pathways, all of which malfunction in hyperhomocysteinemia, suggests that EGF-like domains may be preferential sites of homocysteinylation.

Proteoglycans at the bone-implant interface.

The widespread success of clinical implantology stems from bone's ability to form rigid, load-bearing connections to titanium and certain bioactive coatings. Adhesive biomolecules in the extracellular matrix are presumably responsible for much of the strength and stability of these junctures. Histochemical and spectroscopic analyses of retrievals have been supplemented by studies of osteoblastic cells cultured on implant materials and of the adsorption of biomolecules to titanium powder. These data have often been interpreted to suggest that proteoglycans permeate a thin, collagen-free zone at the most intimate contact points with implant surfaces. This conclusion has important implications for the development of surface modifications to enhance osseointegration. The evidence for proteoglycans at the interface, however, is somewhat less than compelling due to the lack of specificity of certain histochemical techniques and to possible sectioning artifacts. With this caveat in mind, we have devised a working model to explain certain observations of implant interfaces in light of the known physical and biological properties of bone proteoglycans. This model proposes that titanium surfaces accelerate osseointegration by causing the rapid degradation of a hyaluronan meshwork formed as part of the wound-healing response. It further suggests that the adhesive strength of the thin, collagen-free zone is provided by a bilayer of decorin proteoglycans held in tight association by their overlapping glycosaminoglycan chains.

TPA-mediated regulation of osteopontin in human malignant glioma cells.

Malignant gliomas are the most common primary intracranial neoplasms in adults and are largely refractory to post-surgical therapy despite intensive therapeutic efforts. Using a number of different brain tumor-derived cell lines we have demonstrated that the mRNA for osteopontin (OPN), which is substantially over-expressed by some tumors in comparison with normal tissues, is preferentially expressed in high grade and metastatic brain tumors compared to low grade brain tumors. One glioma-derived cell line, U105MG, which does not express significant amounts of OPN mRNA, could be induced dose-dependently by the tumor-promoting and PKC-activating phorbol ester, TPA, to over-express OPN mRNA in a PKC-dependent manner. Unexpectedly, treatment of U105MG cells with Ca2+ ionophore (A23187) completely inhibited TPA-mediated induction of OPN while treatment with the intracellular Ca2+ antagonist TMB-8 had no significant effect. Elucidation of regulatory mechanisms for OPN induction in glioma cells should facilitate rational design of novel therapeutics for human malignant gliomas.

Cyclic GMP-dependent protein kinase inhibits osteopontin and thrombospondin production in rat aortic smooth muscle cells.

Vascular lesions resulting from injury are characterized by a thickening of the intima brought about in part through the production of increased amounts of extracellular matrix proteins by the vascular smooth muscle cells (VSMCs). In this study, we tested the hypothesis that cGMP-dependent protein kinase (PKG), an important mediator of NO and cGMP signaling in VSMCs, inhibits the production of two extracellular matrix proteins, osteopontin and thrombospondin, which are involved in the formation of the neointima. VSMCs deficient in PKG were stably transfected with cDNAs encoding either the holoenzyme PKG-Ialpha or the constitutively active catalytic domain of PKG-I in order to directly examine the effects of PKG on osteopontin and thrombospondin production. Cells expressing either of the PKG constructs had dramatically reduced levels of osteopontin and thrombospondin-1 protein compared with control-transfected PKG-deficient cells. PKG transfection also altered the morphology of the VSMCs. These results indicate that PKG may be involved in suppressing extracellular matrix protein expression, which is one important characteristic of synthetic secretory VSMCs. Suppression of these matrix proteins may underlie the effects of NO-cGMP signaling to inhibit VSMC migration and phenotypic modulation.

Phosphorylation of osteopontin by Golgi apparatus casein kinase.

Osteopontin (OPN) is a ubiquitous multiphosphorylated secretory glycoprotein. Twenty-seven phosphorylated serines have been identified in bovine milk OPN (E. S. Sorensen et al. (1995) Protein Sci. 4, 2040-2049). Nineteen of these phosphoacceptor sites are fully conserved in rat OPN, all displaying the consensus for the Golgi apparatus casein kinase, G-CK (S-x-E/Sp). Here we show that rat OPN is indeed phosphorylated more readily than casein itself by G-CK from either rat mammary gland or liver. OPN is also phosphorylated by casein kinases-1 and -2 (CK1, CK2), though less readily than casein. If OPN kinase activities are normalized in terms of casein phosphorylation, OPN phosphorylation rate by G-CK is 78-fold and 19-fold higher than those measured with CK2 and CK1, respectively. These data, in conjunction with the specific location of G-CK to the Golgi apparatus, where CK2 and CK1 are hardly detectable, support the view that G-CK is the main if not the only physiological agent committed to the phosphorylation of OPN.

Calcitriol enhancement of TPA-induced tumorigenic transformation is mediated through vitamin D receptor-dependent and -independent pathways.

We previously showed that 1alpha,25-dihydroxyvitamin D3, calcitriol, enhanced phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced tumorigenic transformation of mouse epidermal JB6 Cl41.5a cells. To determine if calcitriol regulates this enhancement through a nuclear vitamin D receptor (VDR)-dependent or -independent pathway, we used vitamin D analogs which induce biological responses by either of these mechanisms. In JB6 Cl41.5a cells, 1alpha,24-dihydroxy-22-ene-24-cyclopropyl-vitamin D3 (BT), which like calcitriol binds to VDR and regulates transcription, inhibited cell growth, stimulated expression of nonphosphorylated osteopontin (OPN), and enhanced TPA-induced anchorage-independent growth (AIG, an in vitro assay which highly correlates with tumorigenicity of these cells). 25-Hydroxy-16-ene-23-yne-vitamin D3 (AT), which stimulates calcium influx but has low affinity for VDR, had moderate effects on cell growth and expression of OPN. However, it enhanced TPA-induced tumorigenic transformation, though to a lesser extent than BT, thus suggesting that a VDR-independent mechanism is involved. Since 1alpha-hydroxylase activity was detected in JB6 cells, AT could be converted into 1alpha,25-dihydroxy-16-ene-23-yne-vitamin D3 (V), an analog which binds with high affinity to VDR, and could subsequently enhance TPA-induced AIG. To verify whether the VDR-independent pathway is involved in calcitriol enhancement of tumorigenic transformation, two additional VDR-independent analogs, 1alpha,25-dihydroxy-lumisterol3 (JN) and 24R,25-dihydroxyvitamin D3 (AS), were tested. The analog JN, which stimulates calcium transport and cannot be further hydroxylated at 1-carbon position, increased TPA-induced AIG, while AS, which inhibits calcium influx, did not. These studies suggest that a VDR-independent pathway, perhaps stimulation of calcium influx, and a VDR-dependent mechanism, which directly affects transcription, are involved in calcitriol's enhancement of TPA-induced tumorigenic transformation in JB6 Cl41.5a cells.

Effect of surface topography of titanium on surface chemistry and cellular response.

Surface topography plays a critical role in the interaction of dental implants with adjacent tissues. It has been hypothesized that an increase in surface roughness will result in an increase in calcium and phosphorus deposition after immersion in a simulated physiological solution and will increase protein production and calcium uptake by osteoblast-like cells. With the use of a profilometer, titanium samples ground with 600 grit silicon carbide paper were observed to have an average roughness (Ra) value of 0.28 +/- 0.03 micron, whereas titanium samples polished with 0.3 micron Al2O3 exhibited a Ra value of 0.11 +/- 0.01 micron. X-ray photoelectron spectroscopy analyses indicated the presence of calcium, phosphorus, sodium, and chlorine on both surface conditions after immersion in a protein-free physiologic solution. No significant difference in calcium and phosphorus concentrations were observed between the 600 grit or Al2O3 polished titanium samples after immersion in solution. The Ca/P ratio for both 600 grit and Al2O3 polished titanium was in the range of 0.8 to 1.1 after 12 days in solution. The percent protein retained by the rat bone marrow cell layer on both the Al2O3 polished and 600 grit titanium surfaces increased dramatically during the initial 3 days of the study. The 45Ca assays revealed no significant difference in cellular calcification on Al2O3 polished and 600 grit titanium surfaces. For both the Al2O3 polished and 600 grit surfaces, a sharp increase in 45Ca incorporation was observed after 9 days incubation.

Cofactor role for 10-formyldihydrofolic acid.

10-Formyl-7,8-dihydrofolic acid (10-HCO-H2folate) was prepared by controlled air oxidation of 10-formyl-5,6,7,8-tetrahydrofolic acid (10-HCO-H4folate). The UV spectra of the 10-HCO-H2folate preparation has lambda max. 234, 333 nm and lambda min. 301 nm at pH 7.4, and lambda max. 257, 328 nm and lambda min. 229, 307 nm at pH 1. 1H-NMR spectroscopy of 10-HCO-H2folate (in 2H2O; 300 MHz) suggested a pure compound and gave resonances for one formyl group proton, two protons on C-7 and C-9, and no evidence for a C-6 proton, which is consistent with the structure proposed. The spectral properties indicated that the 10-HCO-H2folate preparation is not appreciably contaminated with 10-HCO-H4folate, 5,10-methenyltetrahydrofolic acid (5,10-CH = H4folate) or 10-formylfolic acid (10-HCO-folate). The above data establish that the 10-HCO-H2folate prepared here is authentic. In contrast, a folate with a UV spectrum having lambda max. 272 nm and lambda min. 256 nm at pH 7, which was prepared by 2,6-dichloro-indophenol oxidation of 10-HCO-H4folate and reported to be 97% pure [Baram, Chabner, Drake, Fitzhugh, Sholar and Allegra (1988) J. Biol. Chem. 263, 7105-7111], is apparently not 10-HCO-H2folate. 10-HCO-H2folate is utilized by Jurkat-cell (human T-cell leukaemia) and chicken liver aminoimidazolecarboxamide ribonucleotide transformylase (AICAR T'ase; EC 2.1.2.3) in the presence of excess 5-amino-imidazole-4-carboxamide ribotide (AICAR) resulting in the appearance of approximately 1 mol of H2folate product for each mol of AICAR formylated. The present 10-HCO-H2folate preparation had a kinetic advantage over 10-HCO-H4folate resulting from a difference of approx. 5-fold in K(m) values when both folates were used as cofactors for Jurkat-cell and rat bone marrow AICAR T'ase. No substantial kinetic advantage was observed using chicken liver AICAR T'ase. 10-HCO-H2folate had little or no activity with Jurkat-cell or chicken liver glycinamide ribonucleotide transformylase (GAR T'ase, EC 2.1.2.2). The existence in vivo of 10-HCO-H2folate is suggested in mammals by several reports of detectable amounts of radiolabelled 10-HCO-folate in bile and urine after administration of radiolabelled folic acid.

Cellular response to well-characterized calcium phosphate coatings and titanium surfaces in vitro.

The cellular response to well-characterized Ca-P and Ti surfaces was analyzed. This study indicated that the ion-beam sputtering process could produce a hydroxyapatite-type coating. However, structural alterations such as the incorporation of CO3 and the loss of the OH groups were observed. The Ti surfaces were shown to be predominantly amorphous TiO2. The in vitro cell culture study showed no significant differences in cell number for the Ti and Ca-P surfaces throughout the 12-day study. Furthermore, the initial percentage of protein within the cell layers was significantly lower for cells on Ca-P surfaces compared with the cell layer on Ti surfaces. However, after 3 h incubation, no significant difference in the percentage of protein was observed for the Ti and Ca-P surfaces.

Osteopontin adhesion receptors on gingival fibroblasts.

Osteopontin (OPN) promotes attachment and spreading of cells in an RGD dependent fashion, suggesting that OPN interacts with integrins on cell surfaces. Here in, we show that LM-609, a monoclonal antibody to the alpha v beta 3 integrin (a vitronectin receptor), inhibited OPN-mediated attachment of gingival fibroblasts. To characterize the cell surface receptors responsible for this interaction, we performed OPN-sepharose affinity chromatography using detergent extracts of 35S-methionine or 125I-surface labeled gingival fibroblasts. Proteins bound to the OPN-matrix were eluted with EDTA and subjected to SDS-PAGE under reducing conditions. EDTA eluates from both 125I-surface labeled and 35S-methionine labeled extracts demonstrated prominent bands in the 90kDa and 50kDa regions, by both autoradiography and fluorography, respectively. These studies suggest that OPN is associated with other cell surface molecules in addition to alpha v beta 3. Furthermore, these as yet to be characterized proteins, may prove to have a stronger affinity for OPN than alpha v beta 3.

Calcitriol regulation of osteopontin expression in mouse epidermal cells.

We previously showed that calcitriol (1 alpha,25-dihydroxyvitamin D3) induces clonal mouse epidermal JB6 Cl41.5a cells to synthesize and secrete a nonphosphorylated form of osteopontin (OPN) in a dose-dependent and metabolite-specific manner. To investigate whether OPN expression is transcriptionally regulated by calcitriol in these cells, we first determined the early time course of calcitriol-induced expression of OPN protein and steady state levels of OPN messenger RNA (mRNA). Calcitriol treatment of JB6 Cl41.5a cells for 6 h caused increased secretion of [35S]methionine-labeled OPN, with maximal levels attained after 8 h of treatment. Northern analyses showed that steady state levels of OPN mRNA increase before the synthesis and secretion of OPN protein. Treatment of JB6 Cl41.5a cells with calcitriol and the transcriptional inhibitor actinomycin-D (2-250 ng/ml) indicated that calcitriol-induced accumulation of steady state OPN mRNA and secretion of OPN protein were dose dependently inhibited by actinomycin-D. These data suggest that calcitriol regulates the expression of OPN at the level of transcription. Furthermore, calcitriol increased the steady state level of OPN mRNA in a dose-dependent manner. Calcitriol-mediated increases in OPN expression were also observed in a transfection assay using a construct consisting of a portion of the promoter region of the OPN gene fused to the luciferase reporter gene. In addition, a study using 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, an adenosine analog that inhibits mRNA synthesis, showed that calcitriol treatment did not significantly increase the stability of OPN mRNA. These findings suggest that calcitriol increases the expression of OPN mRNA and protein by stimulating transcription.

Calcium-binding properties of osteopontin derived from non-osteogenic sources.

Osteopontin (OP), purified from rat bone, binds Ca2+ but whether different molecular forms of OPs derived from non-osteogenic sources and non-phosphorylated OP also possess this property remains to be determined. Furthermore, it is not known which specific site or sites of the molecule bind Ca2+. In the present study, following an established procedure, total proteins in the conditioned media from OP-synthesizing cell cultures were separated by SDS-PAGE, transferred to Immobilon-P membranes, and incubated with 45CaCl2, then Ca2+ ions bound to protein bands were analyzed by autoradiography. Purified OPs, and synthetic oligopeptides representing specific domains of the OP molecule were adsorbed on the membrane and processed as described above. Our results show that OPs synthesized by normal rat kidney cells, oncogenically transformed Rat-1 cells, OP purified from human milk, and non-phosphorylated OP secreted by 1 alpha, 25-dihydroxyvitamin D3-treated mouse epidermal JB6 cells all bind detectable levels of Ca2+ with specificity. We also show that a synthetic peptide representing the domain of OP which contains nine consecutive aspartic acid residues binds Ca2+ with specificity. It is probable, therefore, that a Ca(2+)-binding site resides in this region of the OP molecule. We conclude that Ca(2+)-binding is a general property of OP, irrespective of its molecular mass and origin, and the phosphate moieties of OP may not influence the conformation or accessibility of the Ca2+ affinity sites of the molecule.

Osteopontin-hydroxyapatite interactions in vitro: inhibition of hydroxyapatite formation and growth in a gelatin-gel.

Osteopontin is a phosphorylated bone matrix sialoprotein, postulated to play a regulatory role in biomineralization. The effects of a crude preparation of rat bone osteopontin and a more highly purified bovine bone osteopontin were evaluated using a gel diffusion system to measure effects of 0.1-100 micrograms/ml of this matrix protein on hydroxyapatite formation and crystal proliferation. Bovine osteopontin at concentrations greater than 25 micrograms/ml inhibited both hydroxyapatite formation and growth in a dose-dependent manner. Osteopontin at concentrations lower than 25 micrograms/ml had no detectable effect on the amount of mineral accumulated in experiments with and without pre-formed hydroxyapatite seed crystals either when initial mineral deposition was assessed at 3.5 days, or when mineral formation and growth were assessed at 5 days. There was a statistically significant dose-dependent decrease in crystal length at all concentrations tested. The rat osteopontin preparation had similar inhibitory abilities. Partial dephosphorylation of bovine osteopontin with alkaline phosphatase removed its inhibitory ability, and reduced its ability to bind calcium. The affinity of bovine osteopontin for hydroxyapatite was determined based on a Langmuir adsorption isotherm, with values of K (binding affinity) and N (number of binding sites) being 0.026 ml/microgram and 1084 micrograms/m2, respectively. The data suggest that, in this system, osteopontin is an effective inhibitor of hydroxyapatite formation and growth due to its affinity for the hydroxyapatite crystals. In this system, osteopontin, distinct from other phosphoproteins which both promote and inhibit hydroxyapatite deposition, did not enhance mineral formation at any concentration tested.

1 alpha,25-Dihydroxyvitamin D3 enhances 12-O-tetradecanoylphorbol-13-acetate- induced tumorigenic transformation and osteopontin expression in mouse JB6 epidermal cells.

To study the role of 1 alpha,25-dihydroxyvitamin D3 (calcitriol) in tumor promotion, we used JB6 C141.5a cells, a mouse epidermal cell model of tumor promotion. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) irreversibly induces anchorage-independent growth and tumorigenicity in these cells. Since we previously showed that calcitriol does not transform these cells but inhibits their proliferation, we hypothesized that calcitriol would inhibit TPA-induced transformation. Concurrent treatment of JB6 C141.5a cells with TPA and calcitriol revealed that calcitriol enhanced (1.7- to 10-fold, depending on dose) TPA-induced anchorage-independent growth without enhancing cell proliferation. Furthermore, a more than additive effect on osteopontin mRNA and protein levels was observed with concurrent drug treatment, which yielded a more highly phosphorylated form of osteopontin. These studies suggest coordinate regulation between the signaling pathways for calcitriol and TPA in JB6 C141.5a cells and further implicate expression of phosphorylated osteopontin in tumorigenesis.

Adhesion of metastatic, ras-transformed NIH 3T3 cells to osteopontin, fibronectin, and laminin.

We previously reported that H-ras-induced metastatic ability in murine NIH 3T3 cells is accompanied by increased expression of osteopontin (OPN). OPN is a secreted phosphoprotein that contains a GRGDS amino acid sequence, suggesting adhesive function, but the function of OPN in tumor cells remains poorly understood. Here we report that PAP2 cells (ras-transformed, metastatic NIH 3T3 cells) adhere and spread on OPN-coated substrates, while NIH 3T3 cells adhere and spread poorly on OPN. A similar pattern was seen for adhesion to laminin, while both cell lines adhered equally well to fibronectin. Adhesive interactions to OPN, laminin, and fibronectin were specific and were blocked by GRGDS (but not control GRGESP) peptides. The kinetics of adhesion to all three substrates was examined. Maximum adhesion was observed at 30-60 min, with reduced adhesion thereafter. We also purified metabolically labeled [32P]OPN secreted by PAP2 cells. Labeled OPN bound better in solution to PAP2 cells than to NIH 3T3 cells, and binding to both cell lines was blocked by GRGDS peptides, results that are consistent with the adhesion and spreading of these cells to OPN-coated substrates. Malignant PAP2 cells thus not only secrete increased levels of OPN, relative to NIH 3T3 cells, but also adhere better to this protein. While the target of OPN secreted by tumor cells is not known, our results raise the possibility that tumor cells that secrete OPN may also bind this protein and that this binding may function in autocrine-type signal transduction important to malignancy.