Shedding of growth hormone-binding protein is inhibited by hydroxamic acid-based protease inhibitors: proposed mechanism of activation of growth hormone-binding protein secretase.
The present study describes events postulated to be involved in the regulated mechanism of proteolytic shedding of growth hormone (GH)-binding protein (GHBP). Using Chinese hamster ovary (CHO) cell lines stably transfected either with the full-length human GH receptor (hGHR) or with the cytoplasmic domain-truncated hGHR (hGHR(tr)), we show that the phorbol ester, phorbol 12-myristate 13-acetate (PMA), caused a rapid time- and dose-dependent increase in GHBP secretion, which, as expected, was matched by a corresponding decrease in cell-surface GHR. Furthermore, PMA equally enhanced GHBP release from CHO/hGHR(tr) cells, suggesting that the cytoplasmic domain of hGHR is not essential for PMA-induced shedding. PMA is known to specifically activate protein kinase C and, indeed, the stimulatory effects of PMA in both cell lines were completely inhibited by the protein kinase inhibitor, staurosporine (100 nM), suggesting that activation of protein kinase C (PKC) may mediate PMA-induced GHBP shedding. Since proteolytic cleavage of several cell-surface proteins was shown to be stimulated by modulators of PKC activity and inhibited by metalloprotease inhibitors, we studied the effects of two hydroxamic acid-based inhibitors of zinc-dependent metalloproteases, BB-3103 and Ro31-9790, on GHBP proteolysis. Pretreatment of CHO/hGHR cells with both these inhibitors reduced PMA-enhanced shedding of GHBP, in a dose-dependent manner, with IC(50) values of approximately 0.41 microM for BB-3103 and approximately 0.97 microM for Ro31-9790. In addition, these inhibitors dose-dependently reduced the shedding enhanced by the sulfhydryl alkylator, N-ethylmaleimide (NEM), with IC(50) values of approximately 0.32 microM and approximately 0.58 microM for BB-3103 and Ro31-9790 respectively. It was of interest to find out that Ro31-9790 acted not only to modulate PMA- or NEM-induced shedding processes, but also markedly reduced the spontaneous, time-dependent accumulation of GHBP released from CHO/hGHR cells growing in serum-containing medium. Taken together, these results suggest that one or more zinc-dependent metalloprotease(s), acting at the cell surface, may be involved in GHBP secretase activity. A scheme is proposed whereby at least part of the regulated maturation and/or activation of the protease activity may involve a cysteine-switch mechanism and/or PKC-dependent phosphorylation. In the long run, specific inhibitors of these processes could be applied in the regulation of GHBP levels and, thus, of GH availability and/or activity.
Amit
T
T
Department of Pharmacology, Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel.
Amit
T
T
Hochberg
Z
Z
Yogev-Falach
M
M
Youdim
M B
MB
Youdim
M B
MB
Barkey
R J
RJ
D000818:Animals; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011986:Receptors, Somatotropin / Q000235:genetics / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection
C108282:BB 3103; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D006877:Hydroxamic Acids; D011986:Receptors, Somatotropin; C097661:Ro 31-9790; D011493:Protein Kinase C; D008666:Metalloendopeptidases; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D005033:Ethylmaleimide; C051815:somatotropin-binding protein