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 regulat ed mechanism of proteolytic shedding of growth hormone (GH)-binding protein (GHBP). Using Chinese hamster ovary (CHO) cell lints stably transfected ei ther with the full-length human GH receptor (hGHR) or with the cytoplasmic domain-truncated hGHR (hGHR(tr)), we show that the phorbol ester. phorbol 1 2-myristate 13-acetate (PMA), caused a rapid time-and dose-dependent increa se in GHBP secretion, which, as expected, was matched by a corresponding de crease 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 activa te protein kinase C and, indeed. the stimulatory effects of PMA in both cel l lines were completely inhibited by the protein kinase inhibitor, staurosp orine (100 nM), suggesting that activation of protein kinase C (PKC) may me diate 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-310 3 and Ro31-9790, on GHBP proteolysis. Pretreatment of CHO/hGHR cells with b oth these inhibitors reduced PMA-enhanced shedding of GHBP, in a dose-depen dent manner, with IC50, values of similar to0.41 muM for BB-3103 and simila r to0.97 muM for Ro31-9790. In addition. these inhibitors dose-dependently reduced the shedding enhanced by the sulfhydryl alkylator. N-ethylmaleimide (NEM), with IC50 values of similar to0.32 muM and similar to0.58 muM for B B-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, bu t also markedly reduced the spontaneous, time-dependent accumulation of GHB P released from CHO/hGHR cells growing in serum-containing medium. Taken to gether, these results suggest that orle or more zinc-dependent metalloprote ase(s), acting at the cell surface, may be involved in GHBP secretase activ ity. A scheme is proposed whereby at least part of the regulated maturation and/or activation of the protease activity may involve a cysteine-switch m echanism and/or PKC-dependent phosphorylation. In the long run, specific in hibitors of these processes could be applied in the regulation of GHBP leve ls and, thus, of GH availability and/or activity.