Osteoblasts Selectively Induce Osteopontin Expression in Response to Adhesion on Different Extracelluar Matrix Proteins

Relevance to Muscoloskelatal Conditions Integrin receptors play a structural role in cell adhesion and mediate signal transduction from the extracellular matrix to the cells, thereby modulating gene expression. These functions are crucial in the regulation of bone cell responsiveness to mechanical signals and provide positional information to the cells relative to the extracellular matrix (ECM). Furthermore, these cell functions are crucial in promoting the integration of tissue-engineered scaffolds and biomaterials into the body. Introduction Bone cells play an important role in the structural maintenance and integrity of skeletal tissues, partly through their interactions with the specialized ECM proteins of the bone tissue. These interactions are mediated through receptors (i.e. integrins) that bind to the various proteins of the ECM. Such receptor interactions with the matrix provide positional information to the cells. Thus integrin ligands may act as autocrine and/or paracrine factors in spatially regulating cellular function. In bone tissue, osteopontin (OPN) is one of the predominant RGD-containing integrin ligands of the ECM. OPN is ubiquitously expressed in all skeletal tissues during embryogenesis, and it is localized in areas adjacent to resorptive osteoclasts. This suggests that this protein has an important role in anchoring osteoclasts to bone, allowing for the resorption process. It has been speculated that OPN may be important in the regulation of osteoclast function. Thus, while it is made by osteoblasts, this protein regulates osteoclast function. Our experiments were designed to examine whether osteoblasts would selectively adhere to different ECM ligands, which would then mediate the selective induction of OPN mRNA expression. The hypothesis of these studies is that osteoblasts discriminate between various integrin ligands at a molecular level by using different ECM “ligands” to promote cellular adhesion. Downstream from cellular adhesion is the induction of the selective activation of specific signal transdution processes and the expression of other specific integrin ligands such as osteopontin. Thus the regulation and expression of these proteins act like an autocrine loop to promote the regulation of specific cellular activities. Materials and Methods Cell Culture: Seventeen-day embryonic chicken calvaria were dissected, and osteoblasts were isolated by trypsin collagenase digestion. Attachment Assays: Dishes were coated with 2 μg/cm or 10 μg/cm of Fibronectin (FN), vitronectin (VN), laminin (LN), denatured collagen (i.e. gelatin) (G), collagen type I (Col1), osteopontin (OPN), polyL-lysine (PL) or bovine serum albumin (Al). Values were calculated as a percentage of the cells on the experimental surfaces relative to the cell numbers observed on the plates coated with PL. Signal Transduction Studies: General protein kinase C (PKC) and protein kinase A (PKA) activities were measured using specific fluorescent substrates. Enzyme activities were first normalized per total protein content used in each assay. Values were then calculated as the percentage of activities seen in cells plated on polyL-lysine coated dishes. Immunohistochemical Studies: The intracellular distribution of focal adhesion kinase (FAK), phosphotyrosine (PT) and vinculin (VN) were examined after adhesion on surfaces coated with either PL or FN following standard immunocytochemistry techniques. RNA Analysis: Northern blots were performed with cDNA for OPN. All analyses were performed at least three times, and all data is presented as a percentage in expression over that of the controls determined from parallel cultures. All data were evaluated as the mean ± 1 standard deviation with a minimum of three experiments from different populations of primary cells. Results FN, Col1 and G promoted selective adhesion, while VN, LN and OPN did not. The attachment to FN showed induction of both PKA and PKC activities, while Col1 showed the strongest induction of PKA activity with no induction of PKC activity. In addition, OPN also stimulated PKA activity, contrasting to G and LN, which both strongly inhibited PKA activity. Since FN was shown to be the most effective attachment ligand, this protein was selected to mediate cell adhesion, after which the distribution of focal adhesion kinase (FAK), phosphotyrosine (PT) and vinculin (VN) were examined. These data showed that while adhesion to FN did not appear to alter the general intracellular localization or distribution of FAK or VN within one-hour period, there was a strong induction of PT levels and a generalization of its distribution throughout the cells after adhesion was promoted with a specific binding to FN. At 4 hours, only cells that had been plated onto FN and C showed induction of OPN mRNA expression, which continued to increase up to 24 hours after plating. At 24 hours, cells that had been plated onto LN also showed an increase in OPN mRNA expression. In contrast, adherence surfaces coated with G, OPN and VN did not induce OPN mRNA. These data suggest that those proteins (except for G) that failed to facilitate specific cell adhesion also failed to induce OPN gene expression. Taken together, these results suggest that the specific receptors that promote the selective adherence of osteoblasts also selectively induce the expression of OPN through specific signal transduction mechanisms. Discussion The experiments presented here define how osteoblasts discriminate ECM-mediated signals at a molecular level. In this study we have investigated whether specific cell surface receptors that interact with ECM matrix proteins (i.e. native collagen, osteopontin, fibronectin, denatured collagen, vitronectin and laminin) use common mechanisms in the regulation of the cellular response to adhesion. The ECM is an important structural scaffold for the organization of living tissues in the body, thus providing an array of regulatory signals for numerous cellular functions. The resultant process of osteoblast-ECM adhesion generates an abundance of signals designed to modulate vital osteoblastic responses. Transduction of signals from the ECM takes place through a variety of transmembrane proteins, including the integrin family of cell receptors. Overall, adhesion of osteoblasts appeared to be transduced primarily through integrin receptors. While these results indicate that osteoblast adhesion is specifically promoted by β1 and not by β3 containing integrins, these data are suggestive of a further discrimination between the individual integrin ligands, as LN did not strongly mediate cell adhesion. ECM protein-receptor specificity is an integral part of cellular signal transduction, providing the means for initiation of second messenger signaling cascades. These results show that specific effects on kinase activities are dependent on the selective ligand interactions, which are distinct from those of cell adhesion alone. Similarly, osteoblast adhesion on any of the surfaces studied did not appear to alter the general intracellular localization of FAK or VN. In contrast, adhesion caused a strong induction of proteins containing phosphotyrosine. This was accompanied by a generalized change in the distribution of these proteins from the cell surface to the cytoplasm. Finally, we investigated the molecular activation of intracellular signaling by looking at the osteoblast-ECM adhesion-mediated signals through their regulation of the expression of opn. These results suggest that the same integrin receptors that facilitated specific cell attachment also mediated the induction of OPN expression. It is interesting to note that neither OPN nor VN appeared to promote cell adhesion or induce OPN expression. This suggests that these responses are not facilitated through a αvβ3 receptor. The data taken as a whole suggests that induction of intracellular second signal kinase activity is related to the specific nature of the ligand’s interactions with the receptor, and the processes of cellular-mediated adhesion overlap in some manner with the generation of second signals, which mediate osteopontin indcution.