Isolation and somatomedin activity of bovine growth hormone fragment 87-124.

Various bovine growth hormone (GH) fragments were prepared and tested for somatomedin-like activity in vitro. Cyanogen bromide cleavage followed by reduction and alkylation yielded three fragments which were identified as GH (6-124), GH (150-179) and GH (125-149). No consistent effect was found when these preparations were tested for their ability to stimulate in vitro sulfate and thymidine uptake by rat costal cartilage and to compete with [125I]iodoinsulin for insulin-binding sites on placenta membranes. A fourth peptide was isolated by cleavage of the tryptophanyl and methionly bonds of bovine Gh using anhydrous heptofluorobutyric acid and cyanogen bromide. In addition to significant amounts of non-specific cleavage products, a peptide have a molecular weight of about 4800 was isolated. The amino terminal residue was leucine and the carboxyl terminal was homoserine. These data, in addition to the amino acid composition, suggested that the peptide corresponded to residues 87-124. Fragment GH (87-124) stimulated sulfate (minimum effective concentration, 5 . 10-8 M) and thymidine (minimum effective concentration, 10-8M) uptake by rat costal cartilage. It also cross-reacted, albeit weakly, with insulin-binding sites on placenta membrane. Maximum displacement was 35% of non-specific binding. These observations demonstrate that somatomedin-like activity can be generated from the growth hormone molecule which is inherently devoid of such activity.

Somatomedin-like bioactivities of a growth hormone fragment on embryonic chick cartilage and cultured human fibroblasts.

Somatomedins are growth hormone-dependent peptides which appear to mediate many of the effects of growth hormone in vivo. These peptides are commonly assayed by their enhancement of proteoglycan sulfation in cartilage (or fibroblasts). We now report that fragment A-II (bovine growth hormone, 96--133) stimulates sulfation in chick embryonic cartilage and cultured fibroblasts. Enhancement of fibroblast proteoglycan sulfation by fragment A-II was log-dose dependent with maximal stimulation at 10(-8) M. The 25--100% maximal enhancement by fragment A-II was similar to that reported with a preparation of somatomedin A (Wasteson, A., Uthne, K., Westermark, B. (1973) Biochem. J. 136, 1069--1074). Sulfation of chick cartilage, in the presence of both serum (hypopituitary human) and fragment A-II was greater than the sum of the effects of each substance tested separately. Fragment A-II was tested between 10(-12) and 10(-8) M; maximal stimulation occurred at 5 . 10(-11) M. To our knowledge, no other growth hormone fragment has yet been shown to possess these somatomedin-like bioactivities. Our results suggest that fragment A-II may be very useful as a model peptide to study the actions and mechanism of naturally occurring sometomedins.

Post-receptor actions of somatomedin on chondrocyte collagen biosynthesis.

The anabolic effects of human somatomedin C on freshly isolated Swarm chondrosarcoma chondrocytes were studied. Physiological concentrations (2-34 nM) of somatomedin, but not growth hormone, stimulated the incorporation of radiolabeled precursors into collagen and non-collagen proteins, RNA and proteoglycans. The 120% increase in radiolabeling of collagen with [3H]proline by 10 nM somatomedin C was not due to changes in the rate of [3H]proline uptake. Similarly, the specific activity of newly synthesized [3H]proline-labeled collagen was not increased by 10 nM somatomedin C, indicating that changes in proline pool size or compartmentalization of [3H]proline were not involved. Somatomedin C had no effect on the rate of [3H]collagen degradation. The ability of somatomedin C to stimulate collagen synthesis was unaffected by concentrations of actinomycin D which inhibited chondrocyte RNA synthesis 90% or more. These results demonstrate that somatomedin exerts a general anabolic effect on chondrosarcoma chondrocyte metabolism. The fact that somatomedin C stimulates chondrocyte collagen synthesis in the absence of RNA synthesis suggests that this effect may be at the post-transcriptional level.

Enhancement of cartilage protease activity during age and growth hormone-dependent growth.

We wish to report an intriguing relationship between cartilage protease activity and rat growth rate. This was demonstrated by comparing protease activities of rats having different growth rates, i.e., normal rats of different ages, hypophysectomized and growth hormone-treated hypophysectomized rats. Protease activity, assessed by hydrolysis of a gelatin film by cartilage microtome slices, at pH 4.0, was time and temperature dependent. Preincubation of cartilage tissue at various temperatures resulted in an increase of protease activity from 4 degrees C to 37 degrees C and a decrease in activity from 37 degrees C to 100 degrees C. The activity of younger (4 week old) more rapidly growing rats, was greater than that of older, less rapidly growing animals. Hypophysectomy reduced protease activity to approximately one-third normal levels. However, injection of bovine growth hormone into hypophysectomized rats restored the activity. These results suggest that a positive correlation exists between cartilage protease activity and growth rate. Our results support the novel hypothesis that cartilage growth could be mediated, at least in part, via growth hormone-dependent proteolytic activity.

Nb2 cell mitogenesis: effect of lactogens on cAMP and protein phosphorylation.

Rat lymphoma cells (Nb2) are exquisitely sensitive to lactogenic hormones and are an ideal system to study receptor-mediated signal transduction. The effect of human growth hormone (hGH) on macromolecular synthesis, intracellular cAMP concentrations and protein phosphorylation was investigated in Nb2 cells maintained in serum-free medium. hGH stimulated the incorporation of radiolabeled precursors into protein, RNA and DNA in a time-dependent manner. The concentration of hGH inducing half-maximal DNA synthesis was 11 pM, indicating that Nb2 cells cultured in serum-free medium maintain the same sensitivity to lactogen as cells in horse serum-containing medium. hGH over a period of 4 h had no effect on intracellular cAMP regardless of the presence or absence of isobutylmethylxanthine (IBMX). IBMX (250 microM), increased intracellular cAMP levels 2-fold indicating that the cAMP assay was sufficiently sensitive to detect relatively small changes in intracellular cAMP. Cyclic AMP had no effect on protein phosphorylation. However, hGH, prolactin and placental lactogen enhanced phosphorylation of many protein targets, as well as that of a specific protein (Mr = 29,000). Rat growth hormone, which is not mitogenic, had no effect on protein phosphorylation. These results suggest that lactogen-mediated Nb2 mitogenesis does not involve modulation of intracellular cAMP concentration and that cAMP-independent protein phosphorylation may play a role.

Stimulation of human fibroblast protein, ribonucleic acid, and deoxyribonucleic acid synthesis by bovine growth hormone fragments.

Bovine GH and two fragments which were obtained by dissociation of a limited tryptic digest of the hormone stimulated protein, RNA, and DNA synthesis of contact-inhibited human fibroblasts. The stimulation of protein, RNA, and DNA synthesis was similar for the test substances. Maximal stimulation was noted at 10 nM. At that concentration, protein, RNA, and DNA synthesis were respectively increased 1.80, 1.42, and 1.37 times by GH; 1.93, 1.27, and 1.46 times by A-I (the larger fragment); and 1.99, 1.26, and 1.33 times by A-II (the smaller fragment). The action of GH, A-I, and A-II was similar to that of fetal calf serum, but was distinguished by the time course of stimulation and by the magnitude of the response. For example, GH, A-I, and A-II produced their earliest detectable effect at 10 h for protein synthesis, 22 h for RNA synthesis, and 26 h for DNA synthesis. On the other hand, serum stimulated protein, RNA, and DNA synthesis at 6, 10, and 16 h, respectively. These data show that human fibroblasts respond equally to GH, A-I, and A-II and suggest that there may be more than one "active site" in the GH molecule. Lastly, human fibroblasts may represent a useful system to study the actions of GH in vitro.

Regulation of cartilage acid hydrolases by growth hormone.

The in vivo regulation of three acid hydrolases, namely cathepsin D, cathepsin B, and acid phosphatase, by GH was investigated. The costal cartilage cathepsin D and acid phosphatase activities of hypophysectomized rats were reduced relative to those found in normal controls. Treatment of hypophysectomized animals with GH enhanced rat growth rate and increased these two enzyme activities toward normal levels. Results of pepstatin experiments suggested that the elevated cartilage cathepsin D activity corresponded to an increase in enzyme concentration. A degree of specificity in this regulation was apparent because cartilage cathepsin B, unlike cathepsin D and acid phosphatase, was refractory to hypophysectomy and GH treatment. In contradistinction to cartilage, none of these hepatic enzymes responded to GH, and only cathepsin B activity was diminished by hypophysectomy. Centrifugational and detergent studies indicated that changes in enzyme activities induced by GH treatment were not due to the differential release of acid hydrolases from subcellular compartments. Overall, our results suggest that costal cartilage cathepsin D and acid phosphatase activities are GH dependent and may be related to cartilage growth. These observations may provide insight into the mechanism of GH action and, derivatively, skeletal growth.

Bovine growth hormone fragment (1-133) has in-vitro somatomedin-like activity.

Thrombin digestion of bovine growth hormone (1-191) resulted in cleavage of the peptide bond between amino acid residues 133 and 134. Native growth hormone and purified peptides (1-133) and (134-191) were assayed for somatomedin-like activity. Peptide (1-133), ranging in concentration from 0.15-15 nmol/l, stimulated in-vitro uptake of [3H]thymidine by rat costal cartilage. None of the other peptides was biologically active.

Alteration of Cartilage Lysosomal Enzyme Activities during Development: (acid hydrolases/cartilage development/cartilage growth/hypophysectomy).

Cartilage cathepsin D, cathepsin B and acid phosphatase activities decreased with maturation of Sprague-Dawley rats. Although this phenomenon may largely be due to an age-dependent decrease in cell concentration at young ages (1-8 weeks), in older (8-25 weeks) rats there appeared to be a decrease in enzyme activity per cell. The dimunition in cartilage cathepsin D activity coincided with an apparent decrease in its concentration. In addition, the inverse correlation between rat age and cartilage lysosomal enzyme activities was, at least in part, tissue specific as the pattern of liver lysosomal enzyme activities was quite different from that noted with cartilage. Interestingly, hypophysectomy greatly diminished age-related modulations in lysosomal enzyme activities suggesting that one or more pituitary hormones may be involved in the mechanism of this age-dependent phenomenon. In addition, cartilage growth rate appeared to be correlated with the level of cartilage lysosomal enzyme activities, indicating that these enzymes may be related to the biochemical mechanism of cartilage growth and development.

pN collagen type III within tendon grafts used for anterior cruciate ligament reconstruction.

This study measured the amount of immature collagen type III present in tendon rafts obtained from anterior cruciate ligament (ACL) reconstructions. These values were compared with those obtained from control grafts typically used for reconstruction--Achilles, patellar, and fascia lata--and also to the normal ACL. Analyses were performed using a commercially available radioimmunoassay (RIA). The RIA made use of a rabbit polyclonal antibody specific to the amino terminus of procollagen type III. The specificity of the Ab was confirmed by a western blot. Fibril diameter of each of the above samples was measured by transmission electron microscopy (TEM). We thus were able to determine if there was a relationship between pN collagen III content and fibril diameter. The mean amount of pN collagen type III in the normal tendon control group was 0.8 +/- 0.3 ng/microg total protein (range 0.0-2.5 ng/microg). There was significantly greater pN collagen III (16 +/- 3.7 ng/microg total protein) in the grafts containing an average fibril diameter <55 nm than in the normal tendons or ACL (P < 0.05). Grafts with an average fibril diameter >55 nm had similar levels of pN collagen III (1.0 +/- 0.79 ng/microg) as the controls. There was also significantly less pN-collagen III within the functional grafts (5.3 +/- 1.9 ng/microg) as compared to failed grafts, (21.6 +/- 5.1 ng/microg, P < 0.05). These results indicate that incomplete processing of procollagen III may be responsible for some of the ultrastructural alterations seen in tendon grafts. Since ultrastructural organization is believed to influence mechanical properties of these tissues. pN collagen III levels may be a possible indicator of ligament or tendon weakness.