Cloning and regulation of rat tissue inhibitor of metalloproteinases-2 in osteoblastic cells

Arch Biochem Biophys. 1994 Jun;311(2):313-20. doi: 10.1006/abbi.1994.1243.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 8-Bromo Cyclic Adenosine Monophosphate / pharmacology
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Bone Neoplasms
  • Cloning, Molecular
  • DNA, Complementary
  • Gene Expression Regulation, Neoplastic* / drug effects
  • Gene Library
  • Humans
  • Ionomycin / pharmacology
  • Kinetics
  • Metalloendopeptidases / antagonists & inhibitors*
  • Molecular Sequence Data
  • Neoplasm Proteins / biosynthesis
  • Osteoblastoma
  • Osteoblasts / metabolism*
  • Parathyroid Hormone / pharmacology*
  • Protein Biosynthesis*
  • RNA, Messenger / biosynthesis
  • Rats
  • Second Messenger Systems
  • Sequence Homology, Amino Acid
  • Sequence Homology, Nucleic Acid
  • Tetradecanoylphorbol Acetate / pharmacology
  • Tissue Inhibitor of Metalloproteinase-2
  • Transcription, Genetic / drug effects

Substances

  • DNA, Complementary
  • Neoplasm Proteins
  • Parathyroid Hormone
  • RNA, Messenger
  • Tissue Inhibitor of Metalloproteinase-2
  • 8-Bromo Cyclic Adenosine Monophosphate
  • Ionomycin
  • Metalloendopeptidases
  • Tetradecanoylphorbol Acetate

Associated data

  • GENBANK/U14526