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Biomedical subjects

C Ohlsson

Publications and source records attributed to C Ohlsson.

At least 91 records · Page 5Linked to original sources
PubMed1998

Interleukin-13 inhibits cell proliferation and stimulates interleukin-6 formation in isolated human osteoblasts.

Interleukin-13 (IL-13) is a recently identified cytokine that is secreted by activated T cells and regulates inflammatory responses. We have investigated the effects of IL-13 on isolated human osteoblast-like cells (hOB). IL-13 dose-dependently (1-100 pmol/L) reduced the incorporation rate of [3H]thymidine in hOB cells by more than 50%. Using a cell metabolic assay as well as direct cell counting, we found that treatment with IL-13 lead to a decrease in hOB cell number. The effect was both time and dose dependent, and after 12 days of culture, treatment with IL-13 (0.1 nmol/L) caused a 70% decrease in the number of cells. Also, IL-13 increased the levels of IL-6 messenger ribonucleic acid in hOBs, as measured by ribonuclease protection assay, and stimulated secretion of IL-6 into culture supernatants. In conclusion, IL-13 inhibits cell proliferation and increases IL-6 formation in human osteoblasts. Our findings suggest that IL-13 may cause bone loss due to impaired osteoblastic growth and IL-6-induced osteoclast recruitment.

Cell Division↗
PubMed1998

Treatment with the oral growth hormone secretagogue MK-677 increases markers of bone formation and bone resorption in obese young males.

The effect of 2 months of treatment with the oral growth hormone (GH) secretagogue MK-677 on markers of bone metabolism was determined in healthy obese male subjects. This was a randomized, double-blind, parallel, placebo-controlled study. Twenty-four healthy obese males, 19-49 years of age, with body mass index > 30 kg/m2 were treated with MK-677 (25 mg/day; n = 12) or placebo (n = 12) for 8 weeks. MK-677 increased markers of bone formation; a 23% increase in the carboxy-terminal propeptide of type I procollagen levels and a 28% increase in procollagen III peptide levels were seen with as little as 2 weeks of MK-677 treatment (p < 0.01 and p = 0.001 vs. placebo, respectively) while a 15% increase in serum levels of osteocalcin was not detected until 8 weeks of treatment (p < 0.01 vs. placebo). Markers of bone resorption were induced within 2 weeks of treatment with MK-677; serum levels of the carboxy-terminal cross-linked telopeptide of type I collagen were increased 26% at 8 weeks (p = 0.001 vs. placebo), and urine hydroxyproline/creatinine and calcium/creatinine ratios at 8 weeks were increased by 23% (p < 0.05 vs. placebo) and 46% (p < 0.05 vs placebo), respectively, MK-677 increased serum insulin-like growth factor binding protein-5 (IGFBP-5) by 43-44% after 2-8 weeks of treatment (p < 0.01 vs. placebo). Serum IGFBP-4 was increased by 25% after 2 weeks of treatment (p < 0.001 vs. placebo) but no significant change from baseline was observed after 8 weeks of treatment. Plasma interleukin-6 was not significantly changed by active treatment. In conclusion, short-term treatment of healthy obese male volunteers with the GH secretagogue MK-677 increases markers of both bone resorption and formation. Large increases in serum levels of IGF-1 and IGFBP-5 and a transient increase in serum IGFBP-4 were found. Future long-term studies are needed to investigate if prolonged treatment with MK-677 increases bone mass.

Administration, Oral↗
PubMed1998

Cortisol increases growth hormone-receptor expression in human osteoblast-like cells.

It is well known that high levels of glucocorticoids cause osteoporosis and that physiologic levels of growth hormone (GH) are required for normal bone remodeling. It has been suggested that glucocorticoids regulate GH-responses via the regulation of GH-receptor expression. The aim of the present study was to investigate whether cortisol plays a role in the regulation of GH-receptor expression in cultured human osteoblasts. The effect of serum starvation and cortisol on GH-receptor expression was tested in human osteoblast (hOB)-like cells. Serum starvation for 24 h resulted in an increase in GH-receptor mRNA levels (90 +/- 1% over control culture). Cortisol increased GH-receptor mRNA levels in a dose-dependent manner with a maximal effect at 10(-6)M. The stimulating effect of cortisol on GH-receptor mRNA levels was time-dependent, reaching a peak 12 h after the addition of cortisol (126 +/- 29% over control culture) and remaining up to 12 h later. The increase in GH-receptor mRNA levels was accompanied by an increase in 125I-GH binding which reached a maximum at 24 h (196 +/- 87% over control culture). In conclusion, glucocorticoids increase GH-receptor expression in hOB-like cells. Further studies are needed to clarify whether glucocorticoid-induced regulation of the GH-receptor is important in human bone physiology.

Adolescent↗
PubMed1998

Effects of cortisol on the expression of interleukin-6 and interleukin-1 beta in human osteoblast-like cells.

High levels of glucocorticoids are believed to alter bone remodeling by decreasing bone formation and increasing bone resorption. It has been suggested that different cytokines, like interleukin-6 (IL-6) and interleukin-1 (IL-1), are involved in bone resorption by activating immature osteoclasts, and some studies indicate that IL-6 promotes bone formation by a mitogenic effect on osteoblasts. The aim of the present investigation was to study whether cortisol regulates the expression of IL-6 and IL-1 beta in human osteoblast-like cells. A high dose of cortisol (10(-7)M) decreased, as expected, the C-terminal propeptide of type I collagen released into the culture medium. The IL-6 mRNA levels and IL-6 protein released into the culture medium were also decreased by cortisol in a dose-dependent manner. The maximum effect was seen at 1 microM cortisol (mRNA 23.1 +/- 7.9% of control culture; protein 28.2 +/- 8.3% of control culture). The decrease in IL-6 mRNA levels was apparent 4 h after the addition of cortisol and was still present 20 h later. The decrease in IL-6 protein released into the culture medium was seen 20 h later than the decrease in IL-6 mRNA levels. The production of IL-1 beta protein released into the culture medium was decreased in a dose-dependent manner after the addition of cortisol with a maximum effect at 1 microM. The effect of cortisol on IL-1 beta protein released into the culture medium was seen 16 h after the addition of cortisol. To summarize, cortisol decreases the expression of IL-6 as well as IL-1 beta in human osteoblast-like cells.

Cells, Cultured↗
PubMed1998

Osteoprotegerin mRNA is expressed in primary human osteoblast-like cells: down-regulation by glucocorticoids.

Osteoprotegerin (OPG) is a recently cloned member of the tumour necrosis factor receptor family. It has been suggested that this secreted glycoprotein acts as an inhibitor of osteoclastic differentiation. Expression of OPG has previously been demonstrated in a number of tissues. However, it is still unclear whether or not OPG is expressed by human osteoblasts. We have used the RNase protection assay to demonstrate the OPG transcript in primary cultured human osteoblast-like cells, human marrow stroma cells and osteosarcoma cell lines. Furthermore, we have studied the effect of glucocorticoids on OPG mRNA levels in these cells. We demonstrate that glucocorticoids decrease the OPG transcript in a dose- and time-dependent manner. The time-course study reveals that hydrocortisone (10(-6) M) decreases OPG mRNA levels within 2 h. This decrease is transient, reaching control levels again after 24 h. Our findings demonstrate that human osteoblasts express the mRNA corresponding to OPG, an inhibitor of osteoclast differentiation. The finding that OPG mRNA levels are decreased by glucocorticoids indicates that a reduced production of OPG from osteoblasts and/or marrow stroma cells could, in part, explain glucocorticoid-induced bone resorption.

Anti-Inflammatory Agents↗
PubMed1997

Activation of the prolactin receptor but not the growth hormone receptor is important for induction of mammary tumors in transgenic mice.

Transgenic mice overexpressing the human growth hormone gene develop mammary carcinomas. Since human growth hormone gene can activate both the growth hormone receptor (GHR) and the prolactin (PRL) receptor (PRLR), it is not clear which receptor system is responsible for the malignant transformation. To clarify the receptor specificity, we created transgenic mice with two different genes: (a) transgenic mice overexpressing the bovine growth hormone (bGH) gene having high levels of bGH only activating the GHR and also high serum levels of IGF-I; and (b) transgenic mice overexpressing the rat PRL (rPRL) gene that have elevated levels of PRL (one line 150 ng/ml and one line 13 ng/ml) only binding to the PRLR and with normal IGF-I levels. When analyzed histologically, all of the PRL transgenic female mice developed mammary carcinomas at 11-15 mo of age. Only normal mammary tissue was observed among the bGH transgenic animals and the controls. Cell lines established from a tumor produced rPRL and expressed PRLR. In organ culture experiments, an auto/paracrine effect of rPRL was demonstrated. In conclusion, activation of the PRLR is sufficient for induction of mammary carcinomas in mice, while activation of the GHR is not sufficient for mammary tumor formation.

Adenocarcinoma↗
PubMed1997

Estrogen enhances growth hormone receptor expression and growth hormone action in rat osteosarcoma cells and human osteoblast-like cells.

Postmenopausal bone loss is primarily due to estrogen deficiency. Recent clinical observation demonstrate that GH increases bone mass in GH deficient patients. The present study investigates whether estrogen regulates GH action and GH receptor expression in osteoblasts. 17 beta-estradiol or GH added to the culture medium as single substances did not influence rat osteosarcoma cell proliferation nor human osteoblast-like (hOB) cell proliferation. However, together they synergistically induced osteoblast proliferation (rat osteosarcoma cells 160.1 +/- 15.5% of control cells; human osteoblast-like cells 159.6 +/- 5.1% of control cells). 17 beta-estradiol stimulated 125I-GH binding and GH receptor (GHR) mRNA levels in rat osteosarcoma cells. The stimulatory effect of estradiol was time dependent, reaching a peak after 8 h of incubation with 17 beta-estradiol (binding 216.9 +/- 27.8% and mRNA 374.6 +/- 30.8% of control). The finding that estradiol stimulated 125I-GH binding was confirmed in human osteoblast-like cells. In these cells, 17 beta-estradiol (10(-12) M) increased 125I-GH binding to 203.8 +/- 3.6% of control levels. We conclude that estrogen stimulates GH activity as well as GH binding and GHR mRNA levels in osteoblasts. These findings indicate that estrogen potentiates the effect of GH at the receptor level.

Animals↗
PubMed1997

The CCAAT/enhancer-binding protein-alpha is expressed in the germinal layer of the growth plate: colocalisation with the growth hormone receptor.

The transcription factor C/EBP alpha, a member of the CCAAT/enhancer-binding protein family, is highly expressed in the liver and in adipose tissue. The aim of this study was to determine if C/EBP alpha is expressed in rat growth cartilage. The expression pattern of C/EBP alpha in monolayer-cultured growth plate chondrocytes was similar to that of C/EBP alpha during hepatocyte and preadipocyte differentiation. Immunohistochemistry with a polyclonal antibody for C/EBP alpha revealed that the C/EBP alpha protein is present in the perichondrial ring, in the germinal layer of the growth plate and on the surface of the articular cartilage. The growth hormone (GH) receptor has a similar distribution in the rat tibial growth plate, and hypophysectomised rats were used to investigate a possible connection between C/EBP alpha and GH. C/EBP alpha mRNA levels were decreased in rib cartilage after hypophysectomy. However, GH treatment did not counteract this effect, indicating that other pituitary hormones regulate the C/EBP alpha mRNA levels in growth plate cartilage. We thus demonstrate, for the first time, that C/EBP alpha is expressed in cartilage. The finding that C/EBP alpha, like the GH receptor, is predominantly expressed in stem cell areas of the rat growth plate indicates a possible functional role for C/EBP alpha during early chondrogenic differentiation.

Animals↗
PubMed1996

Rabbit articular cartilage defects treated with autologous cultured chondrocytes.

Adult New Zealand rabbits were used to transplant autologously harvested and in vitro cultured chondrocytes into patellar chondral lesions that had been made previously and were 3 mm in diameter, extending down to the calcified zone. Healing of the defects was assessed by gross examination, light microscope, and histological-histochemical scoring at 8, 12, and 52 weeks. Chondrocyte transplantation significantly increased the amount of newly formed repair tissue compared to the found in control knees in which the lesion was solely covered by a periosteal flap. In another experiment, carbon fiber pads seeded with chondrocytes were used as scaffolds, and repair significantly increased at both 12 and 52 weeks compared to knees in which scaffolds without chondrocytes were implanted. The histologic quality scores of the repair tissue were significantly better in all knees in which defects were treated with chondrocytes compared to knees treated with periosteum alone and better at 52 weeks compared to knees in which defects were treated with carbon scaffolds seeded with chondrocytes. The repair tissue, however, tended to incomplete the bonding to adjacent cartilage. This study shows that isolated autologous articular chondrocytes that have been expanded for 2 weeks in vitro can stimulate the healing phase of chondral lesions. A gradual maturation of the hyalinelike repair with a more pronounced columnarization was noted as late as 1 year after surgery.

Animals↗
PubMed1996

Role of insulin and IGF-I in activation of muscle protein synthesis after oral feeding.

The aim was to evaluate the role of insulin and insulin-like growth factor I (IGF-I) in activation of muscle protein synthesis after oral feeding. Synthesis rate of globular and myofibrillar proteins in muscle tissue was quantified by a flooding dose of radioactive phenylalanine. Muscle tissue expression of IGF-I mRNA was measured. Normal (C57 Bl) and diabetic mice (type I and type II) were subjected to an overnight fast (18 h) with subsequent refeeding procedures for 3 h with either oral chow intake or provision of insulin, IGF-I, glucose, and amino acids. Anti-insulin and anti-IGF-I were provided intraperitoneally before oral refeeding in some experiments. An overnight fast reduced synthesis of both globular (38 +/- 3%) and myofibrillar proteins (54 +/- 3%) in skeletal muscles, which was reversed by oral refeeding. Muscle protein synthesis, after starvation/ refeeding, was proportional and similar to changes in skeletal muscle IGF-I mRNA expression. Diabetic mice responded quantitatively similarly to starvation/refeeding in muscle protein synthesis compared with normal mice (C57 Bl). Both anti-insulin and anti-IGF-I attenuated significantly the stimulation of muscle protein synthesis in response to oral feeding, whereas exogenous provision of either insulin or IGF-I to overnight-starved and freely fed mice did not clearly stimulate protein synthesis in skeletal muscles. Our results support the suggestion that insulin and IGF-I either induce or facilitate the protein synthesis machinery in skeletal muscles rather than exerting a true stimulation of the biosynthetic process during feeding.

Animal Feed↗
PubMed1996

Elevated levels of growth hormone increase bone mineral content in normal young mice, but not in ovariectomized mice.

Both estrogens and GH are necessary for normal bone remodeling. This study investigates the effect of elevated GH levels on the amount and density of bone in young female mice and its dependence on intact ovarian function. Metallothionein promoter-GH-transgenic mice were either sham operated or ovariectomized at 25-29 days of age, and the bone measurements were made at about 90 days of age. A 6-mm high cylinder containing only cortical bone was cut from the right tibia, and lumbar vertebrae 6 was measured as a bone with predominantly cancellous bone. The amounts of tibial and vertebral bone, measured by dry weight, mineral weight, organic weight, bone mineral content (measured by dual energy x-ray analysis), and volume, were increased in GH-transgenic animals compared to those in normal littermates. This stimulatory effect of elevated GH levels was not seen in ovariectomized mice. The real density of the tibial bone were slightly decreased in GH-transgenic animals compared to normal littermates. In conclusion, elevated levels of GH increase the amounts of vertebral (predominantly cancellous) bone and tibial (cortical) bone in young mice. Intact ovaries are a prerequisite for the stimulatory effect of elevated levels of GH. The fact that ovariectomy decreases the stimulatory effect of elevated GH levels suggests that the effect of elevated GH levels in bone is dependent upon the presence of basic sex steroid secretion.

Absorptiometry, Photon↗
PubMed1996

Growth hormone increases interleukin-6 produced by human osteoblast-like cells.

Physiological levels of GH are necessary for normal bone remodeling. GH treatment increases bone resorption and results in enhanced bone formation. The aim of the present study was to investigate whether the GH-induced increase in bone resorption could be mediated by a regulation of interleukin-6 (IL-6) production by human osteoblast-like cells. Cultures of human osteoblast-like cells were established from trabecular bone. Ribonuclease protection analysis demonstrated a high expression of the IL-6 messenger ribonucleic acid (mRNA) transcript in osteoblast-like cells, bone, and muscle, whereas expression was lower in liver. GH increased IL-6 mRNA levels as well as IL-6 protein released into the culture medium from human osteoblast-like cells in a time- and dose-dependent manner, with a maximal effect at 100 micrograms/L (mRNA, 322 +/- 95% of control; protein, 576 +/- 155% of control). In conclusion, GH increases IL-6 produced by human osteoblast-like cells. This in vitro evidence suggests, for the first time, a mechanistic paradigm by which GH modulates bone resorption.

Cells, Cultured↗
PubMed1996

Cortisol decreases IGF-I mRNA levels in human osteoblast-like cells.

Excess levels of glucocorticoids are known to cause osteoporosis. It is speculated that the effect of glucocorticoids could be mediated via regulation of IGF-I. The aims of the present study were to detect and quantify the expression of IGF-I and/or IGF-II mRNA transcripts in human osteoblast-like cells and to investigate whether glucocorticoids regulate the expression of IGF-I mRNA transcripts in human osteoblast-like cells. Cultures of human osteoblast-like cells from trabecular bone were established. The IGF-IA and IGF-IB transcripts were detected in human osteoblast-like cells from seven out of nine patients while the IGF-II transcript was detected in human osteoblast-like cells from eight out of nine patients, as determined by RT-PCR assays. Human osteoblast-like cells, as well as human muscle tissue, expressed approximately 1/10 of the IGF-I mRNA levels found in liver, as determined by RNase protection solution hybridization assay. The IGF-I mRNA levels did not decrease with age in the human osteoblast-like cells and no difference was seen between males and females. However, cortisol (10(-6) mol/l) decreased IGF-I mRNA levels. In summary, the present study has shown that human osteoblast-like cells express IGF-I and IGF-II mRNA transcripts and that cortisol down-regulates the IGF-I mRNA levels, indicating that some of the inhibitory effect of glucocorticoids on bone formation in humans is mediated via a reduced autocrine/paracrine expression of IGF-I.

Adolescent↗
PubMed1996

Growth hormone binds to a single high affinity receptor site on mouse osteoblasts: modulation by retinoic acid and cell differentiation.

Growth hormone (GH) exerts direct differentiative and proliferative effects on osteoblasts. We studied 125I-labeled human (h) GH binding to primary mouse osteoblasts derived from collagenase-treated 18-day fetal mouse calvaria. Scatchard analysis of the data revealed a single class of high affinity GH receptors (apparent Ka = 5.74 x 10(9) M-1) with 2200 sites per cell. Affinity crosslinking and SDS-PAGE electrophoresis showed two bands with apparent molecular masses of 120 and 70 kDa. Mouse osteoblasts express GH receptor mRNA with gene transcripts of 4.2 and 1.2 kb, at levels which reach approximately 1/6 of those in mouse liver and 1/3 of those in mouse muscle. Two populations of undifferentiated and diffentiated osteoblasts, obtained by sequential collagenase digestion of mouse calvaria, were used to study the relationship between osteoblastic phenotype and GH receptor expression. Although the affinity of the receptors in undifferentiated and differentiated cells was the same, the capacity was significantly higher (1.45 +/- 1.0% vs 2.39 +/- 0.9%, P = 0.03) in differentiated cells. This stresses the specific importance of the osteoblast as a target cell for GH. The differentiating potential of the vitamin A derivative retinoic acid was subsequently used experimentally to induce differentiation in the cells. Retinoic acid increased 125I-hGH binding to preosteoblasts (153%, P = 0.02). Together, these data demonstrate the presence of a high affinity GH receptor in mouse osteoblasts which is related to differentiation.

Animals↗
PubMed1996

Growth hormone receptor activity is stimulated by insulin-like growth factor binding protein 5 in rat osteosarcoma cells.

Osteoblast-like UMR-106.01 rat osteosarcoma cells express high affinity growth hormone (GH) receptors (GHRs). Because osteoblasts secrete insulin-like growth factor binding protein-5 (IGFBP-5), we evaluated whether it also modulates GH binding and GHR expression in UMR cells. Human recombinant intact IGFBP-5 stimulated 125I-hGH binding in a dose-dependent manner (dose range 300-3000 ng/ml), inducing an increase to 193.6 +/- 2.1% of control binding at 3000 ng/ml (P < 0.001). Carboxy-truncated IGFBP-5 also stimulated GH binding but with less potency (125 +/- 2.7% of control at 3000 ng/ml, P < 0.01). GHRs identified by chemical crosslinking of 125I-hGH to cell monolayers increased after treatment with IGFBP-5 and decreased in response to insulin-like growth factor-I (IGF-I). GHR mRNA levels, as quantitated by a solution hybridization RNAse protection assay, increased up to 3 to 7-fold in a time-dependent manner by intact IGFBP-5 but not by carboxy-truncated IGFBP-5. An antiserum to IGFBP-5 reduced basal GH binding to 56.7 +/- 4.3% of control value at a concentration of 0.5% (P < 0.001), showing that IGFBP-5 produced by the cells is a strong regulator of GH binding. IGFBP-5 antiserum also decreased GH binding to 85.9 +/- 0.9% of IGFBP-5 stimulated value (P < 0.001), showing the specificity of IGFBP-5 stimulation. To determine whether the GHR upregulation was physiologically significant, cell proliferation was evaluated after coincubation of IGFBP-5 with low, non-stimulatory concentrations of GH. IGFBP-5 (1000 ng/ml) induced cell proliferation to 116.2 +/- 3.2% of control levels, and coincubation with hGH at 10 ng/ml induced an increase to 133.3 +/- 0.1% of control levels. We conclude that exogenous and endogenous IGFBP-5 upregulate GHR mRNA levels and GH binding and this interaction potentiates GH-stimulated mitogenesis in osteoblastic cells.

Animals↗
PubMed1995

The effect of recombinant human IGF-I on protein metabolism in post-operative patients without nutrition compared to effects in experimental animals.

This study has evaluated the effects of recombinant human insulin-like growth factor I (rhIGF-I) to moderately stressed post-operative patients provided with dextrose as the only exogeneous substrate. Thirty patients who underwent elective colorectal surgery were randomized to receive either rhIGF-I (80 micrograms kg-1 bw) subcutaneously twice daily or placebo injections in a double-blind parallel group design. Nitrogen balance, urinary 3-methyl-histidine excretion plasma growth hormone (GH), serum cortisol, IGF-I binding proteins (IGFBP-1,3), glomerular filtration rate, plasma amino acid concentrations and whole-body energy expenditures were measured as effector variables during days 1-5 post-operatively. Animal and isolated tissue experiments were performed as additional control experiments to confirm cellular effectiveness of the recombinant material. rhIGF-I increased significantly the glomerular filtration rate and prevented the adaptive decrease in whole-body energy expenditure in response to partial starvation in the postoperative period. Serum and plasma concentrations of IGFBP-1,3 cortisol, blood glucose and amino acids were not significantly influenced by rhIGF-I administration, while plasma GH levels decreased significantly as expected. rhIGF-I had no effect on either nitrogen balance or protein breakdown (3-methylhistidine excretion) in post-operative patients on dextrose supplementation only, although plasma concentrations of IGF-I increased from 130-140 ng mL-1 to a range of 300-450 ng mL-1. In contrast, IGF-I stimulated the synthesis of both globular and myofibrillar proteins (+50%, P < 0.01), when given as a single dose (100 micrograms kg-1) 2 h before measurements of protein synthesis in skeletal muscles of overnight fasted adult mice. This stimulatory effect by IGF-I (1 microgram mL-1) was also confirmed by measurements of skeletal muscle protein synthesis in vitro (+40%, P < 0.05). Orally re-fed mice had a normal transcription of IGF-I mRNA in skeletal muscle cells, while overnight fasted mice showed a trend to down-regulated transcription. Our results demonstrate that rhIGF-I has several significant physiological effects, without major side-effects, when supplied to partially starved patients in the post-operative phase. The lack of a whole-body nitrogen sparing effect by rhIGF-I alone to post-operative patients is not clear, but was most likely explained by subnormal plasma concentrations of amino acids.

Aged↗
PubMed1995

Insulin-like growth factor binding proteins-2 and -3 stimulate growth hormone receptor binding and mitogenesis in rat osteosarcoma cells.

GH exerts its biological actions on osteoblasts through a specific high affinity receptor expressed on these cells. GH receptor binding is positively modulated by a number of factors, including retinoic acid and dexamethasone, whereas fetal calf serum strongly decreases the binding. To identify responsible factors in serum, components of serum, the insulin-like growth factors (IGFs)-I and -II, and IGF binding proteins (IGFBPs)-2 and -3 were tested for a possible negative modulatory role. IGF-I and -II decreased [125I]hGH binding at an optimal concentration of 30 ng/ml for IGF-I and 100 ng/ml IGF-II, reducing the binding to 51% and 55%, respectively, of control values. A stimulation of [125I]hGH binding was observed with IGFBP-2 as well as IGFBP-3, inducing an increase to 148% and 151% of control binding at an optimal concentration of 3000 ng/ml for both peptides. The effects of all peptides were dependent on the incubation time, being significantly increased after 8 h of incubation and reaching the full effect thereafter. The effects were declined at 24 h compared with 16 h for IGFBP-2 and -3 but not for IGF-I and -II. Coincubation of the cells with IGF-I and -II and IGFBP-2 and -3 neutralized the effects of the factors alone. In conclusion, these results show that IGF-I and -II on the one hand and IGFBP-2 and -3 on the other hand exert opposite actions on [125I]hGH binding, IGFBP-2 and -3 exerting probably an IGF-independent effect. Further, IGF-I and -II decreased GH receptor messenger RNA (mRNA) levels, as quantified by a solution hybridization ribonuclease protection assay, from 8.65 +/- 1.78 attomoles (amol)/microgram DNA (control) to 2.4 +/- 0.68 and 2.16 +/- 0.92 amol/microgram DNA, respectively. IGFBP-2 increased GH receptor mRNA levels from 5.26 +/- 1.17 (control) to 13.19 +/- 3.48. Incubation with IGFBP-3 did not result in stimulation of GH receptor mRNA levels (8.59 +/- 2.91 amol/microgram DNA). This shows that the mechanism of regulation of the GH receptor is, except for IGFBP-3, at least in part on the mRNA level. Lastly, IGFBP-2 and IGFBP-3 are mitogenic for UMR-106.01 rat osteosarcoma cells, inducing an increase in cell number to 125% and 142% of control cell counts after 48 h of incubation with 1000 ng/ml IGFBP-2 and -3, whereas IGF-I, IGF-II and Long R3 IGF-I did not stimulate proliferation. IGFBP-2 and -3 potentiate hGH induced mitogenesis at low hGH concentrations of both factors, whereas at higher concentrations no such effect is observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗