Parathyroid hormone (PTH) plays a central role in regulation of calcium metabolism. For example, excessive or inappropriate production of PTH or the related hormone, parathyroid hormone related protein (PTHrP), accounts for the majority of the causes of hypercalcemia. Both hormones act through the same receptor on the osteoblast to elicit enhanced bone resorption by the osteoclast. Thus, the osteoblast mediates the effect of PTH in the resorption process. In this process, PTH causes a change in osteoblastic gene expression. For instance, in response to PTH, mRNA levels for collagen and osteopontin decline while those for interleukin-6 and collagenase are markedly elevated. We have shown that the increase in collagenase mRNA in UMR 106- ()I cells is due to 80-fold changes in transcription of the gene. The PTH-induced changes in collagenase transcription are cycloheximide- sensitive in the first 2 h of PTH stimulation indicating a need for induced expression of some other gene at this time. In other words, PTH regulation of collagenase is a secondary response, with the primary effect of the hormone on another gene. Correlated to these observations, we have shown PTH elicits 20-fold stimulation of c-fos mRNA and protein in this time interval. Thus, c-fos (a member of the activator protein-I, AP-l, transcriptional factor family) may serve as a pivotal gene in linking PTH-evoked events in the cytoplasm to those in the nucleus. Because the c-fos stimulation is mimicked by cAMP analogs, we hypothesize that PTH affects its transcription by: protein kinase A activation causing phosphorylation and activation of cAMP response element binding (CREB) protein which then induces c-fos transcription. Consequently, the overall goal of this proposal is to delineate the mechanisms in the osteoblast which link PTH interaction with its receptor and generation of second messengers to the immediate regulation of gene expression, in is case c-fos, a likely tertiary messenger in the osteoblast. To do this, we will: l) determine the minimal PTH- responsive element in the c-fos gene and assess whether this includes the CRE 62 bp upstream of the transcriptional start sit , 2) examine the nuclear complexes binding to this PTH-minimal response element and identify if CREB is one of the proteins binding to this element, if the foregoing aims support a role for cAMP and CREB in stimulation of c-fos by PTH, 3) ascertain if CREB is phosphorylated in osteoblastic cells after treatment with the hormone, and if this is so, 4) assess whether this is due to activation of protein kinase A. The results of this work will provide insight into one of the key pathways of regulation of the osteoblast. In so doing, the data will also provide new perspectives into treatment of disorders of calcium metabolism.
Showing the most recent 10 out of 22 publications
