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Interleukin-1 and Phorbol Ester Inhibit Collagen Synthesis in Osteoblastic MC3T3-E1 Cells by a Transcriptional Mechanism

Department of Medicine, University of Connecticut Health Center Farmington, Connecticut 06032
Department of Medicine, Veterans Administration Medical Center Newington, Connecticut 06111

Address requests for reprints to: Dr. John R. Harrison, Department of Medicine AM-047, University of Connecticut Health Center, Farmington, Connecticut 06032.

Abstract

The effects of recombinant human interleukin-1 (IL-1) on procollagen gene expression were examined in the clonal mouse osteoblastic cell line MC3T3-E1. Cells were grown in Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum and 50 µg/ml ascorbic acid. Collagen synthesis was assessed as [³H]proline incorporation into collagenase-digestible protein (CDP). Procollagen mRNA levels were determined by Northern blot analysis using a ³²P-labeled ₁(I) cDNA. Transcription rates were determined by nuclear run-off assay. IL-1 at 1–1000 pg/ml caused a concentration-dependent inhibition of CDP, which was maximally reduced by 75–80%, and a parallel reduction of procollagen ₁(I) mRNA levels. The effects of IL-1 were mimicked by the tumor promoter phorbol 12-myristate 13-acetate (PMA) at 1–100 nM, which inhibited CDP and reduced procollagen ₁(I) mRNA levels to a similar extent. The effects of IL-1 and PMA were independent of prostaglandin production, since indomethacin did not alter the inhibitory effect of either agent on CDP. Neither IL-1 (up to 10 ng/ml) nor PMA (100 nM) affected adenylate cyclase activity, while forskolin (10 µM), PTH (10 µM) and prostaglandin E₂ (1 µM) stimulated adenylate cyclase activity 3- to 5-fold. However, forskolin (10 µM) and (Bu)₂cAMP (100 µM) failed to alter CDP or procollagen ₁(I) mRNA levels. IL-1 (1 ng/ml) and PMA (100 nM) reduced transcription of the ₁(I) procollagen gene by 70% and 80%, respectively, while ₂(I) transcription was decreased by 59% and 53%. Neither IL-1 nor PMA affected transcription of the β-actin or β-tubulin genes. In conclusion, IL-1 inhibits collagen synthesis in MC3T3-E1 cells by a transcriptional mechanism which appears to be independent of cAMP. Since PMA mimics the effects of IL-1 on collagen gene expression, protein kinase-C may mediate these actions of IL-1.

FOOTNOTES

A portion of this work was presented at the 11th Annual Meeting of the American Society for Bone and Mineral Research, September 9–14,1989, Montreal, Canada. This work was supported by Grants AR-29850, AM-18063, and AR-31263 from the NIH and funds from the Medical Research Service of the V.A.

^* Recipient of National Research Service Award AR-08031 from the NIH.

Received for publication August 21, 1989. Revision received November 1, 1989. Accepted for publication November 1, 1989.

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