Cloning and regulation of rat tissue inhibitor of metalloproteinases-2 in osteoblastic cells.
Rat tissue inhibitor of metalloproteinases-2 (TIMP-2) was cloned from a UMR 106-01 rat osteoblastic osteosarcoma cDNA library. The 969-bp full-length clone demonstrates 98 and 86% sequence identity to human TIMP-2 at the amino acid and nucleic acid levels, respectively. Parathyroid hormone (PTH), at 10(-8) M, stimulates an approximately twofold increase in both the 4.2- and 1.0-kb transcripts over basal levels in UMR cells after 24 h of exposure. The PTH stimulation of TIMP-2 transcripts was not affected by the inhibitor of protein synthesis, cycloheximide (10(-5) M), suggesting a primary effect of the hormone. This is in contradistinction to regulation of interstitial collagenase (matrix metalloproteinase-1) by PTH in these same cells. Nuclear run-on assays demonstrate that PTH causes an increase in TIMP-2 transcription that parallels the increase in message levels. Parathyroid hormone, in its stimulation of TIMP-2 mRNA, appears to act through a signal transduction pathway involving protein kinase A (PKA) since the increase in TIMP-2 mRNA is reproduced by treatment with the cAMP analogue, 8-bromo-cAMP (5 x 10(-3) M). The protein kinase C and calcium pathways do not appear to be involved due to the lack of effect of phorbol 12-myristate 13-acetate (2.6 x 10(-6) M) and the calcium ionophore, ionomycin (10(-7) M), on TIMP-2 transcript abundance. In this respect, regulation of TIMP-2 and collagenase in osteoblastic cells by PTH are similar. However, we conclude that since stimulation of TIMP-2 transcription is a primary event, the PKA pathway must be responsible for a direct increase in transcription of this gene.
Cook
T F
TF
Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, Missouri 63104.
Burke
J S
JS
Bergman
K D
KD
Quinn
C O
CO
Jeffrey
J J
JJ
Partridge
N C
NC
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001859:Bone Neoplasms; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D015723:Gene Library; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000096:biosynthesis; D018215:Osteoblastoma; D010006:Osteoblasts / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000494:pharmacology*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D015290:Second Messenger Systems; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019716:Tissue Inhibitor of Metalloproteinase-2; D014158:Transcription, Genetic / Q000187:drug effects
D018076:DNA, Complementary; D009363:Neoplasm Proteins; D010281:Parathyroid Hormone; D012333:RNA, Messenger; D019716:Tissue Inhibitor of Metalloproteinase-2; D015124:8-Bromo Cyclic Adenosine Monophosphate; D015759:Ionomycin; D008666:Metalloendopeptidases; D013755:Tetradecanoylphorbol Acetate