Autocrine regulation of cell proliferation by the insulin-like growth factor (IGF) and IGF binding protein-3 protease system in a human prostate carcinoma cell line (PC-3).

PC-3 cells, whose growth is androgen-independent, were shown to be capable of slow proliferation in serum-free medium and in the absence of added growth factor for 7 days. They secreted insulin-like growth factor (IGF)-II but no detectable IGF-I. This IGF-II, although produced in small amounts, plays a role in their proliferation because growth could be inhibited dose dependently by up to 80% in the presence of monoclonal antibodies directed against IGFs or the type 1 IGF receptor. PC-3 cells also secreted IGF binding proteins (IGFBPs) -2, -3, -4, and -6. Immunoblot analysis revealed selective proteolysis of IGFBP-3, yielding fragments of the same molecular size as those generated from IGFBP-3 in vivo. With the addition to the culture medium of a serine protease inhibitor, 4-(2-aminoethyl)-benzenesulfonyl fluoride (Pefabloc-SC), at concentrations < 0.2 mM that were nontoxic to the cells, cell proliferation was dose dependently inhibited up to 80% and, at the same time, proteolysis of the IGFBP-3 secreted by the cells was depressed. Urokinase activity detected in the conditioned media was depressed by Pefabloc, suggesting that the urokinase-type plasminogen activator was involved in the proteolysis of IGFBP-3. In addition, 0.01-5 micrograms/ml plasminogen induced a dose-dependent increase in both proliferation and the proportions of proteolysed IGFBP-3 in the media. The stimulation of proliferation was totally blocked in the presence of anti-type 1 IGF receptor antibody. Recombinant human IGF-II (5-200 ng/ml) added to cell-free medium conditioned by 48 h of culture dose dependently stimulated PC-3 cell proliferation. At concentrations < or = 100 ng/ml, its mitogenic action was potentiated when medium had been conditioned by cells cultured in the presence of plasminogen but inhibited when medium had been conditioned by cells cultured in the presence of Pefabloc. We conclude from these results 1) that IGF-II is involved in the autocrine control of PC-3 cell proliferation via the type 1 IGF receptor; and 2) that this proliferation is directly dependent on IGF-II bioavailability that itself is modulated by the limited IGFBP-3 proteolysis induced, at least in part, by urokinase-type plasminogen activator and plasmin.

Lupus nephritis in a child with AIDS.

Concomitant acquired immunodeficiency syndrome (AIDS) and lupus nephritis is an exceptional feature in white patients. A white boy with maternofetal human immunodeficiency virus (HIV) infection had no medical follow-up until he presented at 12 years of age with a nephrotic syndrome, macrohematuria, renal failure, pancytopenia, and low CD4(+) cell count. A renal biopsy revealed severe lupus nephritis (World Health Organization class IV) with specific immune deposits in the absence of any clinical sign of systemic lupus erythematosus or specific autoantibodies at the time of diagnosis. The treatment consisted of methylprednisolone pulses followed by oral prednisone; antiretroviral triple therapy was started a few weeks later, which contributed to clinical and biologic improvement. To our knowledge, this is the first case report of lupus-like nephritis in a white child with AIDS, whose outcome might be improved significantly by a combination of steroids and antiretroviral therapy.

Prostate carcinoma (PC-3) cell proliferation is stimulated by the 22-25-kDa proteolytic fragment (1-160) and inhibited by the 16-kDa fragment (1-95) of recombinant human insulin-like growth factor binding protein-3.

In a previous study, the biphasic effect of increasing dosages of recombinant human insulin-like growth factor binding protein-3 (rhIGFBP-3) on proliferation in the prostate carcinoma PC-3 cell line (stimulation followed by depression) was shown to reflect changes in the bioavailability of IGF-II secreted by the cells, IGF-II being the major factor responsible for their autocrine growth. These changes depend on the extent of IGFBP-3 proteolysis induced by serine proteases, in particular, plasmin. In order to examine the mechanism of action of IGFBP-3, we investigated the effects of its two major fragments isolated by HPLC following limited proteolysis by plasmin in vitro. The predominant fragment with an apparent molecular mass of 22-25 kDa in SDS-PAGE (under non-reducing conditions) had previously been shown to retain weak affinity for IGFs, whereas the other fragment of 16 kDa lost all such affinity. From their recently determined amino acid sequences, these fragments correspond to the first 160 and 95 residues, respectively, of IGFBP-3. 0.5-5 nM intact rhIGFBP-3(1-264), when pre-incubated with 5 nM rhIGF-II, dose-dependently inhibited (up to 100%) its mitogenic effect, via sequestration owing to its strong affinity for IGF-II. The same concentrations of the larger fragment (IGFBP-3(1-160)) elicited only weak inhibition (up to 30%), coherent with its weak affinity. The smaller fragment (IGFBP-3(1-95)) provoked total inhibition despite its lack of affinity for IGFs and therefore by an IGF-independent mechanism. PC-3 cells in serum-free medium were weakly stimulated by 5 nM intact IGFBP-3. This had previously been shown to be related to its proteolysis and the ratio of proteolysed to intact IGFBP-3. At the same concentration, IGFBP-3(1-160) stimulated this proliferation by a factor of 5-7, whereas IGFBP-3(1-95) totally suppressed it. 5 nM IGFBP-3(1-95) inhibited the mitogenic action of 1% fetal calf serum by 80%, but by only 25% in the presence of an antibody blocking the type 1 IGF receptor. Its inhibition is therefore exerted principally, but not exclusively, via the IGF signalling pathway. Our data indicate that the IGFBP-3 fragments composed of residues 1-160 and 1-95 are biologically active on PC-3 cells and that their opposite actions may account for the events observed when IGFBP-3 is proteolysed in the cell environment. These proteolytic fragments may therefore play a role in the development of prostate adenocarcinomas in vivo.

Recombinant human insulin-like growth factor (IGF) binding protein-3 stimulates prostate carcinoma cell proliferation via an IGF-dependent mechanism. Role of serine proteases.

Insulin-like growth factor-binding proteins (IGFBPs) modulate IGF action at cellular level, through either inhibition or potentiation, and they also have intrinsic activity that is independent of their binding to IGFs. In prostate carcinoma (PC-3) cells, which are capable of growth for several days in serum-free medium, non-glycosylated recombinant human IGFBP-3 (rhIGFBP-3) had a biphasic mitogenic effect, stimulation being dose-dependent up to 20 ng/ml, followed by progressive depression down to zero stimulation at 150-200 ng/ml. This mitogenic effect was not intrinsic activity, but involved IGF-II secreted by the cells, since stimulation was abolished in the presence of anti-type 1 IGF receptor antibody (alpha IR-3). Western ligand- and immunoblot analysis of the culture media revealed several IGFBP species, in particular IGFBP-3 which exhibited an electrophoretic profile characteristic of limited proteolysis. The amounts of the proteolytic fragments increased in parallel with the concentrations of added rhIGFBP-3, but a large amount of intact protein remained at the highest concentrations added. When a serine protease inhibitor, 4-(2-aminoethyl)-benzenesulphonyl fluoride (Pefabloc SC), was added at concentrations demonstrated to be non-toxic to the cells, IGFBP-3 proteolysis was diminished and rhIGFBP-3-induced stimulation of proliferation was suppressed. Conversely, in the presence of plasminogen transformed to plasmin by urokinase secreted by the cells, proliferation stimulated by rhIGFBP-3 and its proteolysis were enhanced. Our results suggest that the biphasic mitogenic effect of rhIGFBP-3 on PC-3 cells reflects changes in the availability to the cells of the IGF-II they secrete. This availability depends on the extent of IGFBP-3 proteolysis (which promotes release of bound IGF-II) and on the proportion of intact forms (which sequestrate secreted IGF-II).