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 within the biologically active PTH-(1-34) peptide are responsible for different functions. The first two N-terminal amino acids are required for plasma membrane adenylyl cyclase stimulation, and the C-terminal region 29-32 is necessary for the translocating activity of protein kinase C. In the present study, we explored the structure-function relationship 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 caused 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 protein (-35.3%; P = 0.001) at 10(-7) M (n = 6). hPTH-(1-31) also decreased 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 D3 [1,25-(OH)2D3] led to 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)2D3 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). hPTH-(1-31) also decreased the receptor up-regulatory effect of 1,25-(OH)2D3 from 370% to 286% (P < 0.02) at 10(-8) M and 220% (P < 0.002) at 10(-7) M, respectively (n = 6). hPTH-(3-34) and -(13-34) at concentrations of 10(-9)-10(-7) M did not decrease VDR content in either the absence or presence of 1,25-(OH)2D3. Quantitation of VDR messenger RNA 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), inhibited ROS 17/2.8 cell VDR gene expression in both the absence and presence of 1,25-(OH)2D3.(ABSTRACT TRUNCATED AT 400 WORDS)
Sriussadaporn
S
S
Department of Medicine, University of Chicago Pritzker School of Medicine, Illinois 60637, USA.
Wong
M S
MS
Whitfield
J F
JF
Tembe
V
V
Favus
M J
MJ
D000262:Adenylyl Cyclases / Q000032:analysis / Q000378:metabolism / Q000502:physiology; D000818:Animals; D001483:Base Sequence; D001859:Bone Neoplasms / Q000737:chemistry* / Q000473:pathology / Q000648:ultrastructure; D002117:Calcitriol / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D015342:DNA Probes / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000737:chemistry* / Q000473:pathology / Q000648:ultrastructure; D012516:Osteosarcoma / Q000737:chemistry* / Q000473:pathology / Q000648:ultrastructure; D010281:Parathyroid Hormone / Q000737:chemistry / Q000494:pharmacology / Q000502:physiology*; D010703:Phorbol Esters / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011493:Protein Kinase C / Q000502:physiology; D051381:Rats; D018167:Receptors, Calcitriol / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D010281:Parathyroid Hormone; D010703:Phorbol Esters; D018167:Receptors, Calcitriol; D005576:Colforsin; D011493:Protein Kinase C; D000262:Adenylyl Cyclases; D002117:Calcitriol