Parathyroid hormone (hPTH 1-38) stimulates the expression of UBP41, an ubiquitin-specific protease, in bone.
Parathyroid hormone (PTH) stimulates bone formation in both animals and humans, and the expression of a number of genes has been implicated in the mediation of this effect. To discover new bone factors that initiate and support this phenomenon, we used differential display reverse transcription polymerase chain reaction (DDRT-PCR) and screened for genes, which are differentially expressed in osteoblast-enriched femoral metaphyseal primary spongiosa of young male rats after a single subcutaneous (s.c.) injection of hPTH (1-38) (8 microg/100 g). We found and cloned one full-length cDNA, which encodes a putative 348 amino acid protein. Sequence analysis of this protein demonstrates a 98, 93.7, and 82.5% identity with mouse, human, and chicken ubiquitin-specific protease UBP41, respectively. Northern blot analysis confirmed that a 3.8-4 kb UBP41 mRNA transcript was rapidly increased 1 h after acute hPTH (1-38) exposure in both metaphyseal (6- to 8-fold) and diaphyseal (3-fold) bone, but returned to control levels by 24 h after exposure. In contrast, continuous exposure to hPTH (1-38), resulted in a rapid and sustained elevation of UBP41 mRNA. PTH (1-31), which stimulates intracellular cAMP, and PTHrP (1-34) both induced UBP41 mRNA expression; whereas PTH analogs (3-34) and (7-34), that do not stimulate cAMP, had no effect on UBP41 expression. UBP41 mRNA expression was also rapidly induced 1 h after injection of PGE2, but returned to the control level by 6 to 24 h. In vitro, UBP41 mRNA is expressed in primary osteoblasts (metaphyseal and diaphyseal derived) and in the osteoblast-like cell lines UMR106, ROS17/2.8, and BALC. PTH (1-38) treatment induced UPB41 expression (3.6- to 13-fold) in both primary cultures of osteoblasts and in UMR106 cells. Further analysis in UMR 106 cells demonstrated that PGE2, forskolin and dibutyryl cAMP increased UBP41 mRNA expression 4-, 4.5-, and 2.4-fold, respectively. Tissue distribution analysis of UBP41 mRNA detected transcripts in brain, heart, skeletal muscle, kidney, liver, and testis. Together, these results demonstrate that UBP41, an ubiquitin-specific protease, is selectively upregulated in bone by the osteotropic agents PTH, PTHrP, and PGE2, possibly via the PKA/cAMP pathway. We speculate that the rapid induction of UBP41 in response to these physiological regulators contributes to the mechanism by which either the structure, activity, half-life or localization of essential proteins are modified to maintain bone homeostasis.
Miles
R R
RR
Gene Regulation, Bone and Inflammation Research Division, Lilly Research Labs, Eli Lilly & Co, Indianapolis, Indiana 46285, USA.
Sluka
J P
JP
Halladay
D L
DL
Santerre
R F
RF
Hale
L V
LV
Bloem
L
L
Patanjali
S R
SR
Galvin
R J S
RJ
Ma
L
L
Hock
J M
JM
Onyia
J E
JE
D000818:Animals; D015152:Blotting, Northern; D001842:Bone and Bones / Q000187:drug effects* / Q000378:metabolism; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D010450:Endopeptidases / Q000096:biosynthesis* / Q000235:genetics; D005269:Femur / Q000378:metabolism; D020869:Gene Expression Profiling; D015723:Gene Library; D008297:Male; D010006:Osteoblasts / Q000378:metabolism; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010446:Peptide Fragments / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D025801:Ubiquitin / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase; D015854:Up-Regulation
D017931:DNA Primers; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D012333:RNA, Messenger; D025801:Ubiquitin; C035747:parathyroid hormone (1-38); D010450:Endopeptidases; C108665:USP2 protein, human; D043222:Ubiquitin Thiolesterase