STRUCTURE-FUNCTION RELATIONSHIP OF HUMAN PARATHYROID-HORMONE IN THE REGULATION OF VITAMIN-D-RECEPTOR EXPRESSION IN OSTEOBLAST-LIKE CELLS (ROS-17 2.8)/

Studies of the relationship between PTH structure and function in the activation of protein kinases have revealed that different regions wit hin the biologically active PTH-(1-34) peptide are responsible for dif ferent functions. The first two N-terminal amino acids are required fo r plasma membrane adenylyl cyclase stimulation, and the C-terminal reg ion 29-32 is necessary for the translocating activity of protein kinas e C. In the present study, we explored the structure-function relation ship of human (h) PTH in the regulation of the vitamin D receptor (VDR ) in osteoblast-like cells (ROS 17/2.8). VDR-rich cytosol extract was prepared after the confluent cells were incubated with different hPTH fragments for 16 h. hPTH-(1-34) at concentrations of 10(-9)-10(-7) M c aused a dose-dependent decrease in VDR content from a control level of 70.2 +/- 2.2 fmol/mg protein to 62.1 +/- 3.3 (-16%) at 10(-9) M, 52.3 +/- 5.3 (-25.5%; P < 0.02) at 10(-8) M, and 45.5 +/- 3.5 fmol/mg prot ein (-35.3%; P = 0.001) at 10(-7) M (n = 6). hPTH-(1-31) also decrease d VDR content from 65.5 +/- 3.6 to 55.2 +/- 7.9 (-19.5%) at 10(-9) M, 44.3 +/- 5.8 (-32.4%; P < 0.05) at 10(-8) M, and 40.6 +/- 3.2 fmol/mg protein (-38.9%; P < 0.05) at 10(-7) M (n = 6). Incubation of ROS 17/2 .8 cells with 0.5 nM 1,25-dihydroxyvitamin D-3 [1,25-(OH)(2)D-3] led t o up-regulation of VDR content by 340-370% of the control value. hPTH- (1-34) decreased the VDR up-regulatory effect of 1,25-(OH)(2)D-3 from 340% to 230% of the control value at 10(-8) M (P < 0.0001) and 170% of the control value (P < 0.0001) at 10(-7) M, respectively (n = 6). hPT H-(1-31) also decreased the receptor up-regulatory effect of 1,25-(OH) (2)D-3 from 370% to 286% (P < 0.02) at 10(-8) M and 220% (P < 0.002) a t 10(-7) M, respectively (n = 6). hPTH-(3-34) and -(13-34) at concentr ations of 10(-9)-10(-7) M did not decrease VDR content in either the a bsence or presence of 1,25-(OH)(2)D-3. Quantitation of VDR messenger R NA by reverse transcription-polymerase chain reaction showed that PTH- (1-34) and -(1-31) at 10(-7) M, but not PTH-(3-34) and -(13-34), inhib ited ROS 17/2.8 cell VDR gene expression in both the absence and prese nce of 1,25-(OH)(2)D-3. These observations indicate that the PTH regio n containing the adenylyl cyclase-stimulating domain is responsible fo r PTH-mediated down-regulation of VDR in ROS 17/2.8 cells.