The bone is a dynamic living tissue with many functions, including locomotion, protection of the inner organs, and mineral homeostasis. To fulfill these various functions, bone tissue must be remodeled continuously throughout an organism’s lifetime. In this process, old bone tissue is broken down and new bone tissue is formed. Although largely comprised of mineralized matrix, the bone also cont[Ca²⁺+]_ins several cell types that are responsible for both the formation and m[Ca²⁺+]_intenance of bone. The cells responsible for breaking down bone are called osteoclasts, while the cells responsible for bone formation are called osteoblasts. An osteoblast that becomes trapped in the matrix it secretes terminally differentiates into an osteocyte, which is the most common cell type in the bone.

After osteoclasts resorb bone tissue, osteoblasts must be recruited to the site of resorption in order to form new bone. Osteoblasts are formed from mesenchymal stem cells in the bone marrow. When exposed to the appropriate environment, the stem cell differentiates into an osteoblast progenitor cell and then into a mature osteoblast that secretes osteoid onto the bone surface. This osteoid is later mineralized to form new bone. The development of the mesenchymal stem cell into a mature osteoid-secreting osteoblast is a complex process that is not well understood.

Osteoblast development is marked by a period of rapid cellular proliferation, crucial for expanding the cell population before the osteoblast progenitor differentiates into a mature osteoblast. Calcium (Ca²⁺+) is central to cellular proliferation, and both intracellular calcium release and the entry of Ca²⁺+ from the extracellular environment through channels have been implicated in the processes of proliferation and differentiation. While our lab has shown that the Ltype voltage-sensitive calcium channel (L-VSCC) is essential for the increase in bone mass in response to mechanical load, I postulated that this mechanical response of bone results from the L-VSCC–mediated activation of proliferation of osteoblastic precursor cells.

Proliferation of MC3T3-E1 pre-osteoblastic cells was significantly inhibited when the L-VSCC-specific inhibitor, nifedipine, was added to the growth medium. When extracellular ATP was hydrolyzed with apyrase, proliferation of MC3T3-E1 cells also was reduced to levels similar to those seen with nifedipine, suggesting a link between calcium entry through the L-VSCC and purinergic signaling. Addition of extracellular ATP increased osteoblast proliferation through activation of the calcineurin-NFAT pathway. However, ATP was not able to rescue proliferation of MC3T3-E1 cells exposed to nifedipine, suggesting converging regulation of osteoblast proliferation by the L-VSCC and ATP, rather than a linear pathway. I also demonstrated that L-VSCC activation activates calmodulin and that osteoblast proliferation was dependent on Ca²⁺+/calmodulin-dependent protein kinase II (CaMKII). Taken together, these data suggest that both the activity of the L-VSCC and purinergic signaling activate converging calcium-mediated signaling pathways to regulate pre-osteoblast proliferation.

As these progenitor cells proliferate in the marrow microenvironment, they also attach to their surrounding extracellular matrix (ECM). During bone remodeling, osteoblast adhesion to the ECM is a critical event necessary for proper function of the osteoblast and the response of these cells to hormonal and mechanical stimuli. I postulated that Ca²⁺+ signaling is essential to the regulation of osteoblast adhesion to fibronectin. To delineate the Ca²⁺+ pathways important in adhesion, I used Ca²⁺+ imaging techniques to determine the global intracellular ([Ca²⁺+]i) response of MC3T3-E1 preosteoblastic cells elicited by 5µg/ml fibronectin. Addition of soluble fibronectin to MC3T3-E1 cells produced a slow, sust[Ca²⁺+]_ined increase in [Ca²⁺+]i, whereas the ECM-associated peptide RGDS caused a smaller, more rapid increase in [Ca²⁺+]i. This Ca²⁺+ response to fibronectin is dependent upon both extracellular and intracellular Ca²⁺+. Inhibition of the L-type voltage sensitive Ca²⁺+ channel (L-VSCC) with nifedipine decreased the [Ca²⁺+]i response to soluble fibronectin as compared to controls.

To characterize binding of the pre-osteoblast to fibronectin, I utilized atomic force microscopy (AFM). Using AFM, I determined that inhibition of the LVSCC reduces binding forces between fibronectin and the cell. Attachment of a fibronectin-coated AFM tip to the osteoblast induces a calcium response that is reduced in the absence of L-VSCC activity. Using immunofluorescence to visualize the focal adhesion protein vinculin, I found that treatment of MC3T3-E1 cells with soluble fibronectin during attachment increased focal adhesion formation as compared to untreated cells and that this response was dependent on L-VSCC activity. These data suggest that calcium entry through the L-VSCC mediates attachment of pre-osteoblasts to fibronectin in the marrow cavity adjacent to the bone surface in order to regulate osteoblast recruitment to the bone surface during bone remodeling.

During osteoblast differentiation, osteoblast precursor cells are marked by a proliferative period that is necessary for establishing a critical number of preosteoblasts that differentiate into mature, bone-forming osteoblasts. These preosteoblasts must also interact with their surrounding ECM. If either process of proliferation or attachment is hindered, the formation and m[Ca²⁺+]_intenance of bone is affected adversely. Previous work from our lab has demonstrated the importance of the L-VSCC in mechanical load-induced bone formation. Here I provide evidence that this calcium channel also is essential for the proliferation and attachment of the pre-osteoblast. This work has implications in the treatment of diseases such as osteoporosis that continue to plague our aging population.