J J Van Wyk

Evidence for a functional role of endogenously produced somatomedinlike peptides in the regulation of DNA synthesis in cultured human fibroblasts and porcine smooth muscle cells.

Cultured porcine aortic smooth muscle cells and human fibroblasts produce somatomedinlike peptides and secrete them into the surrounding microenvironment. This production has been linked to their ability to replicate. The objective of this study was to determine if a specific anti-somatomedin-C (Sm-C) monoclonal antibody that binds the somatomedinlike peptides could inhibit replication by porcine aortic smooth muscle cells and human fibroblasts. To determine if the antibody could inhibit the effect of endogenously produced somatomedinlike peptide, increasing concentrations of antibody were co-incubated with platelet-derived growth factor, a known stimulant of somatomedinlike peptide secretion, and Sm-C-deficient platelet-poor plasma. Addition of the antibody reduced fibroblast [3H]thymidine incorporation from 35,100 +/- 500 to 10,600 +/- 700 cpm (P less than 0.001), and in smooth muscle cells from 29,600 +/- 1,800 to 10,800 +/- 1,100 cpm (P less than 0.001). Co-incubation of exogenously added Sm-C (20 ng/ml) with maximally inhibitory dilutions of antibody increased [3H]thymidine incorporation in fibroblasts from 7,800 +/- 1,000 to 18,900 +/- 800 cpm (P less than 0.01), and in smooth muscle cells from 9,800 +/- 1,200 to 17,200 +/- 1,100 cpm (P less than 0.01). Insulin, which can substitute for Sm-C as a mitogen and does not bind to the antibody, stimulated DNA synthesis when co-incubated with the antibody, thereby excluding the possibility of nonspecific cytotoxicity. These results strengthen the hypothesis that the rate of DNA synthesis of these two cell types in vitro is directly linked to their capacity to produce somatomedinlike peptides. They further support the cellular production of somatomedinlike peptides as examples of the autocrine model of growth regulation.

Cultured fibroblast monolayers secrete a protein that alters the cellular binding of somatomedin-C/insulinlike growth factor I.

We studied somatomedin-C/insulinlike growth factor (Sm-C/IGF-I) binding to human fibroblasts in both adherent monolayers and in suspension cultures. The addition of Sm-C/IGF-I in concentrations between 0.5 and 10 ng/ml to monolayers cultures resulted in a paradoxical increase in 125I-Sm-C/IGF-I binding and concentrations between 25 and 300 ng/ml were required to displace the labeled peptide. The addition of unlabeled insulin resulted in no displacement of labeled Sm-C/IGF-I from the adherent cells. When fibroblast suspensions were used Sm-C/IGF-I concentrations between 1 and 10 ng/ml caused displacement, the paradoxical increase in 125I-Sm-C/IGF-I binding was not detected, and insulin displaced 60% of the labeled peptide. Affinity cross-linking to fibroblast monolayers revealed a 43,000-mol wt 125I-Sm-C-binding-protein complex that was not detected after cross-linking to suspended cells. The 43,000-mol wt complex was not detected after cross-linking to smooth muscle cell monolayers, and binding studies showed that 125I-Sm-C/IGF-I was displaced greater than 90% by Sm-C/IGF-I using concentrations between 0.5 and 10 ng/ml. Because fibroblast-conditioned medium contains the 43,000-mol wt complex, smooth muscle cells were incubated with conditioned medium for 24 h prior to initiation of the binding studies. 125I-Sm-C/IGF-I-binding increased 1.6-fold compared to control cultures and after cross-linking the 43,000-mol wt complex could be detected on the smooth muscle cell surface. Human fibroblast monolayers secrete a protein that binds 125I-Sm-C/IGF-I which can be transferred to the smooth muscle cell surface and alters 125I-Sm-C/IGF-I binding.

CLINICAL STUDIES WITH RECOMBINANT-DNA-DERIVED METHIONYL HUMAN GROWTH HORMONE IN GROWTH HORMONE DEFICIENT CHILDREN

Thirty-six children with growth hormone deficiency were treated for up to 48 months with methionyl human growth hormone (hGH) synthesised by DNA recombinant methods. The growth rate for these children increased from 3.2 +/- 1.1 cm/yr to 10.5 +/- 2.2 cm/yr (mean +/- SD). This was similar to the effect of pituitary hGH in ten GH deficient children, 3.8 +/- 1.0 to 10.1 +/- 1.1 cm/yr. Serum somatomedin C rose from 0.26 +/- 0.23 U/ml to 0.79 +/- 0.53 U/ml after 6 months of methionyl-hGH therapy, similar to the effect of pituitary hGH. The incidence of antibody formation to methionyl-hGH was higher than that observed with pituitary hGH (Kabi) but poor growth was observed only in the one patient on methionyl-hGH who acquired high-titre high-binding-capacity antibodies to hGH. No consistent changes in levels of antibodies to Escherichia coli proteins were detected. No other allergic manifestations or systemic side-effects were demonstrable.

Explorations of the Insulinlike and Growth-Promoting Properties of Somatomedin by Membrane Receptor Assays

Publisher Summary This chapter presents the explorations of the insulinlike and growth-promoting properties of somatomedin by membrane receptor assays. A competitive radioreceptor assay using 125 I-labeled insulin and a human placental cell membrane preparation was developed to measure the total insulinlike activity (ILA) during the fractionation of outdated human plasma. After isoelectric focusing, a major peak of ILA focused in a neutral zone and a minor peak in a more basic zone; conversely, more of the bioassayable somatomedin activity focused in the basic zone than in the neutral zone. The basic fraction was further purified by preparative electrophoresis at pH 2.3 on a 15% acrylamide gel containing 4 M urea. After additional chromatographic steps on Sephadex, an arginine-rich peptide was obtained and designated somatomedin C. Somatomedin C labeled with radioactive iodine was found to possess its own primary binding site in human placental cell membranes, chick embryo chondrocyte membranes, and membranes from rat liver, thymus, kidney, muscle, and brain. The somatomedin C receptor was 100 times more sensitive to competitive binding by unlabeled somatomedin than was the insulin receptor and 1000 times less sensitive to competitive inhibition by insulin.

CHANGES IN CIRCULATING SOMATOMEDIN-C LEVELS IN BROMOCRIPTINE-TREATED ACROMEGALY

To determine whether the improvement in clinical status of patients with active acromegaly correlates with a reduction of circulating somatomedin‐C, serum immunoreactive somatomedin‐C was measured in twenty‐seven patients before and during bromocriptine therapy. The patients were assessed using a clinical and metabolic score which included both subjective criteria of improved sweating and facial features, and objective criteria of a reduction in ring circumference and the area under the glucose tolerance curve. Using this, together with the change in GH levels before and during bromocriptine therapy, the patients could be divided into three groups. In one group, there was no clinical improvement, a less than 30% decline in somatomedin‐C, and in four of six patients no significant decline in GH. In both the other groups there was clinical improvement and a greater than 30% decrease in somatomedin‐C. In one of these two groups, however GH did not decline, while in the other it was reduced significantly. The results suggest that during bromocriptine treatment of acromegaly, serum somatomedin‐C concentrations correlate better with clinical status than does serum GH. Since some patients have no significant fall in GH but show both clinical improvement and a reduction in somatomedin‐C, it seems likely that in some patients bromocriptine may improve acromegaly by a mechanism other than a simple decrease in total immunoreactive GH secretion.

Localization of somatomedin-C binding to bovine growth-plate chondrocytes in situ

The somatomedins are a family of low-molecular-weight peptides that are thought to mediate the stimulatory effect of growth hormone on skeletal growth. The cells that are directly responsible for skeletal growth are the chondrocytes of the epiphyseal growth plate, and these are the presumed skeletal target cells for somatomedin. As with other peptide growth factors, the cellular effects of the somatomedins are initiated by the interaction of the growth factor with specific receptors on target-cell surface membranes. Chondrocytes that have been isolated from bovine growth plates were previously found to possess specific surface receptors for the principal growth-hormone-dependent somatomedin, somatomedin-C. These studies indicated that the interaction of growth-plate chondrocytes with somatomedin-C involves specific receptor-binding followed by somatomedin-C internalization by the cell, a process identical to that identified in the mechanism of action of other peptide growth factors in other cells. These studies, however, left unanswered the questions of whether there are differences in binding of somatomedin-C by the different cell populations within the physis and whether somatomedin-C has access to cells in intact tissues. The current studies address these issues and indicate that bovine physeal chondrocytes in situ are accessible to exogenous somatomedin-C, that they specifically bind somatomedin-C in situ, and that cells of different physeal zones bind somatomedin-C differently. Labeled somatomedin-C is specifically bound by cells of all physeal zones. However, the binding is greatest for those cells undergoing active synthesis of DNA in the proliferative zone.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficient pubification of somatomedin-C/insulin-like growth factor I using immunoaffinity chromatography

Somatomedin-C/insulin-like growth factor I was purified from human plasma using a monoclonal antibody affinity column. Combining immunoaffinity chromatography with standard protein purification methods resulted in an overall recovery of 18%. The 35 micrograms of somatomedin-C/insulin-like growth factor I purified from 500 ml of plasma appeared as a single band when analyzed by polyacrylamide gel electrophoresis and could be used in radioimmunoassay and receptor binding studies.

Prophylactic Adrenalectomy of a Three-Year-Old Girl With Congenital Adrenal Hyperplasia: Pre- and Postoperative Studies

Long term follow-up studies of children with congenital adrenal hyperplasia have documented less than desirable outcomes, including reduction in final adult height, obesity, virilism, and decreased fertility. We have proposed that children with the most severe forms of congenital adrenal hyperplasia would be better off if their adrenals were removed at an early age. We report here on our experience with prophylactic bilateral adrenalectomy in a 3-yr-old girl with a double null mutation of the CYP21 gene. The results of sodium balance studies, performed preoperatively on our patient and her unaffected fraternal twin sister, and hormonal data are presented as well. In contrast to her twin, who markedly increased her sodium retention in response to ACTH, our patient showed increased natriuresis, suggesting a deleterious effect of her adrenals on sodium homeostasis. Adrenalectomy was carried out at the time of necessary genital repair. No surgical or postsurgical complications were encountered.

Localization and regulation of IGF-I and IGF-II mRNA

Insulin-like growth factors I and II (IGF-I and -H) are peptide mitogens that exert a wide range of biological actions in many tissues and cell types. The actions of the IGFs include metabolic and differentiative effects as well as their capacity to stimulate cell proliferation. (Humbel 1984; Froesch et al. 1985; Van Wyk 1984). Traditionally the IGFs were considered as hormones that are transported in the circulation to act on target cells in an endocrine fashion (Humbel 1984; Froesch et al. 1985; Van Wyk et al. 1984). Based on studies of perfused liver (Schwander et al. 1983), liver derived cell lines (Moses et al. 1980), and primary cultures oliver cells (Richmond et al. 1985), the liver was considered the major source of serum IGFs. The IGFs do not appear to be stored in the liver to an appreciable extent, however, since the concentrations of IGFs are higher in blood perfusing the liver than in extracts of liver. More recently, cultured expiants of many tissues in addition to liver have been found to secrete immunoreactive IGFs into media (D’Ercole et al. 1984). Extracts of multiple tissues in addition to liver also contain higher concentrations of immunoreactive IGF than can be attributed to concentrations in blood perfusing the tissues (D’Ercole et al. 1984, 1986). These observations have raised the possibility that in addition to the endocrine actions of IGFs transported to target cells via the circulation, there may be paracrine or autocrine actions of IGFs synthesized locally in multiple tissues (D’Ercole et al. 1984,1986).

Structural similarity of human and bovine somatomedin receptors and human insulin receptor: Analysis by affinity labeling

The growth promoting and insulin-like actions of somatomedin-C (Sm-C) are thought to be mediated respectively by interactions with the Sm-C and insulin (In) receptors. Since Sm-C and In are structurally homologous, we compared the size and subunit structure of their respective radiolabeled receptors in human placental membranes (hPM) and bovine chondrocytes (bC) by crosslinking with disuccinimidyl suberate (DSS). The labeled hormone-receptor complexes (RCs) were analyzed by SDS polyacrylamide gel electrophoresis (PAGE) and autoradiography. Specificity of the respective RCs was demonstrated by competitive inhibition studies. Both hPM RCs had apparent molecular weights of > 250,000 daltons. Reduction with 2-mercaptoethanol yielded subunits of ~ 140,000 daltons. Limited proteolysis with trypsin, chymotrypsin and staphylococcal V-8 protease of the labeled RCs gave similar products. The Sm-C RC In bC isolated from calf epiphyseal plates also contained a 140,000 dalton binding subunit indistinguishable from the HPM receptor. Conclusions: 1) the hPM receptor for Sm-C has a 140,000 MW binding subunit linked by disulfide bonds. 2) By this technique, the hPM Sm-C receptor is structurally indistinguishable from either the Sm-C receptor in bC or the In receptor in hPMs, 3) The sizes of In and Sm-C receptors and their subunits differ from that reported for MSA and certain other hormones.