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 fu ll-length clone demonstrates 98 and 86% sequence identity to human TIM P-2 at the amino acid and nucleic acid levels, respectively. Parathyro id hormone (PTH), at 10(-8) M, stimulates an approximately twofold inc rease 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 transcript s was not affected by the inhibitor of protein synthesis, cycloheximid e (10(-5) M), suggesting a primary effect of the hormone. This is in c ontradistinction to regulation of interstitial collagenase (matrix met alloproteinase-l) by PTH in these same cells. Nuclear run-on assays de monstrate that PTH causes an increase in TIMP-2 transcription that par allels the increase in message levels. Parathyroid hormone, in its sti mulation of TIMP-2 mRNA, appears to act through a signal transduction pathway involving protein kinase A (PRA) 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 l2-myristate 13-a cetate (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 primar y event, the PRA pathway must be responsible for a direct increase in transcription of this gene. (C) 1994 Academic Press, Ins.