UCSF DIABETES, ENDOCRINOLOGY & METABOLISM TRAINING PROGRAM FACULTY RESEARCH SUMMARIES

Calcium-sensing and Calcium Receptors

My laboratory works on the signal transduction, cell biology, and biochemical properties of extracellular calcium-sensing receptors (CaRs). Specifically, we are trying to understand how cells sense changes in the extracellular calcium concentration ([Ca] e ) and then respond with changes in cell function -- that change either rates of hormone secretion such as that of parathyroid hormone (PTH) or change cell differentiation and growth properties.

We are working with cultured mouse bone marrow cells and with mouse growth plate chondrocytes in this work at present. In murine bone marrow cells, we are investigating the effects of Ca and other agonists of the CaR on the differentiation and function of cells early in the osteoblastic and osteoclastic cell lineages. To this end, we are in the process of finishing a series of studies to assess the effects of growing unfractionated bone marrow cells at different [Ca]. We are looking at changes in gene expression, protein levels for certain key osteoblastic markers, and tartrate resistant acid phosphatase (TRAP) and alkaline phosphatase (AP) activities. We are determining whether CaRs are expressed in unfractionated bone marrow cells and or in cells fractionated by magnetic activated cell sorting (MACS) using MAbs directed against markers for bone marrow stromal cells (cells early in the osteoblastic lineage) – VCAM-1 (vascular cell adhesion molecule-1) and for macrophage/monocyte lineage cells (CD11b). The hypothesis underlying these studies is that extracellular Ca -- generated via the resorption of bone matrix -- serves as a signal to stimulate osteoblasts and their precursors to reverse the resorptive process and stimulate bone formation. This would serve to maintain “coupling” between bone resorption and formation that maintain the balance of bone remodeling.

To date, our studies argue strongly that high [Ca] – similar to what might be generated in vivo with a potent resorptive stimulus -- instead of stimulating formation, increases the expression of the receptor activator of nuclear factor kappa B ligand (RANKL) mRNA and protein. The interaction between RANKL and its receptor RANK would then promote osteoclastogenesis. We have shown that osteoclastogenesis does in fact occur in these cultures, grown at high [Ca] – by demonstrating the expression of genes specific for osteoclasts (TRAP, calcitonin receptors, RANK etc) and by the presence of large/multinucleated TRAP positive cells. CaRs are also strongly expressed in the VCAM-1 positive cells.

These studies challenge a previously held paradigm in this area of investigation -- that locally generated Ca from the resorption of bone matrix serves as a locally produced “coupling factor” to reverse the resorptive process. Our data are more compatible with the idea that locally generated Ca, in fact, serves as part of a pathologic “feed-forward” mechanism that could explain why pathologic bone resorption is not down-regulated by local mechanisms. Studies are in progress to test the possible role of CaRs in specific bone marrow cell populations, that we will target in transgenic mice, with either dominant negative or constitutively active CaRs using available type 1 collagen or TRAP promoters.

My laboratory is also assessing the role of extracellular Ca and CaRs on promoting the differentiation of cells in the growth plate in collaboration with Dr. Wenhan Chang. We have found that Ca and CaRs appear to play an important role in modulating the pace of growth plate chondrocyte differentiation in culture. Specifically low [Ca] retard and high [Ca] accelerate the expression of cartilage marker genes and the elaboration of a mature extracellular matrix. We have further found that extracellular Ca and the potent cartilage differentiation molecule PTH-related protein (PTHrP) work in concert to maintain a steady pace of chondrocyte differentiation in vitro. We are further investigating the interaction between the extracellular [Ca] and the growth hormone/insulin-like growth factor 1 system. High [Ca] stimulate increased expression of IGF-1 and its receptor suggesting that the pro-differentiation effects of high [Ca] could be modulated at least in part by the IGF-1 signaling pathway. Studies are underway to understand the underlying mechanisms.

Selected References

Loretz CA, Pollina C, Hyodo S, Chang W, Takei Y, Shoback D. cDNA cloning and functional expression of a Ca 2+ -sensing receptor with truncated intracellular domain from the Mozambique tilapia. J Biol Chem 279: 53288-97, 2004.

Chang W, Shoback D. Extracellular Ca 2+ -sensing receptors - an overview. Cell Calcium 35: 183-196, 2004.

Rodriguez L, Tu C, Cheng Z, Chen T-H, Oda Y, Bikle D, Shoback D, Chang W. Expression and functional assessment of an alternatively spliced extracellular Ca 2+ -sensing receptor in growth plate chondrocytes. Endocrinology 146(12):5294-303, 2005

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