Haplotype insufficiency for suppressor of cytokine signaling-2 enhances intestinal growth and promotes polyp formation in growth hormone-transgenic mice

GH may improve intestinal growth or function in patients with short bowel syndrome. Excessive trophic effects of GH or IGF-I may contribute to neoplastic growth or increased colorectal cancer risk in acromegaly. Identification of mechanisms that limit the tumorigenic potential of GH and IGF-I is desirable. Suppressor of cytokine signaling-2 (SOCS2) limits GH action on body and organ growth, but its role in GH action on intestine is unknown. We tested the hypothesis that SOCS2 limits GH-induced intestinal growth or neoplasia in vivo. GH-transgenic (GH-TG) mice were crossed with SOCS2 null mice to generate wild-type (WT) or transgenic (TG) mice with zero (HO-WT; HO-TG), one (HT-WT; HT-TG), or two (WT-WT; WT-TG) functional SOCS2 genes. No HO-TG mice were derived from crossbreeding. WT-WT, HT-WT, WT-TG, and HT-TG were compared. Body weight, small intestine and colon growth, and levels of jejunal IGF-I and sucrase-isomaltase mRNAs were assessed. Colon was analyzed for abnormal lesions. HT-WT did not differ from WT-WT. Compared with WT-TG, HT-TG had significantly increased body weight, small intestine growth, and local IGF-I expression and decreased sucrase-isomaltase expression. HT-TG colon spontaneously developed multiple hyperplastic and lymphoid polyps. GH-induced activation of STAT5 DNA binding activity was enhanced in intestine of SOCS2 null mice compared with WT control. Haplotype insufficiency for SOCS2 promotes trophic actions of GH in small intestine and promotes preneoplastic growth in colon during excess GH. Small variations in SOCS2 expression levels may significantly influence the outcome of therapeutic GH or acromegaly in intestine.     

Differential expression of suppressors of cytokine signalling genes in response to nutrition and growth hormone in the septic rat

GH treatment during critical illness and sepsis may increase mortality. A family of negative regulators of cytokine signalling, the suppressors of cytokine signalling (SOCS), have been characterised. SOCS provide a mechanism for cross-talk between the cytokine receptors, including GH. Here, we have investigated the impact of nutrition and GH treatment on GH receptor, SOCS1, SOCS-2, SOCS-3 and cytokine-inducible SH2-containing protein (CIS) hepatic mRNA expression in a rat model of sepsis, caecal ligation and puncture (CLP). Four groups of rats were studied: control (food given ad libitum, n=7), CLP only (n=8), CLP and total parenteral nutrition (TPN) (n=9), and CLP, TPN and GH (n=10). CLP rats underwent surgery and 18 h later received saline or TPN or TPN+GH for 6 h before they were killed. Serum IGF-I levels were lower in all CLP groups (P<0.001). The combination of TPN and GH treatment increased IGF-I levels compared with the saline-treated CLP rats (P<0.01), but IGF-I levels remained lower than control animals (P<0.001). GH receptor and GH-binding protein expression in liver was reduced in animals subjected to CLP and was unaffected by nutrition or GH treatment. Hepatic SOCS-1 was detectable in normal rats, induced in all CLP animals but was unaffected by nutrition and GH. Hepatic SOCS-2 expression was difficult to detect in normal and CLP rats but was greatly induced in CLP rats treated with GH. Hepatic SOCS-3 expression was only just detectable in the control group but was elevated in all CLP groups and unaffected by nutrition and GH. Hepatic CIS expression was difficult to detect in normal rats, was not induced by CLP but was induced by both nutrition and GH. In conclusion, CLP induced low IGF-I levels associated with increased expression of SOCS-1 and SOCS-3, both of which are known to inhibit GH receptor signalling. GH induced SOCS-2 and CIS in the CLP rat despite resistance with respect to IGF-I generation, and parenteral feeding induced CIS in the CLP rat. Thus, there is potential for a complex interaction between GH and cytokine signalling at the level of SOCS expression whereby the inflammatory response may alter GH signalling and GH may influence the inflammatory response.     

Evidence supporting dual, IGF-I-independent and IGF-I-dependent, roles for GH in promoting longitudinal bone growth

The possibility that growth hormone (GH) has effects on long bone growth independent of insulin-like growth factor-I (IGF-I) has long been debated. If this is true, then long bone growth should be more profoundly affected by the absence of GH (since both GH and GH-stimulated IGF-I effects are absent) than by the absence of IGF-I alone (since GH is still present and actually elevated). To test this hypothesis, we compared long bone growth in mice with targeted deletions of Igf1 vs growth hormone receptor (Ghr). Tibial linear growth rate was reduced by approximately 35% in Igf1 null mice and by about 65% in Ghr null mice between postnatal days 20 and 40, a time of peak GH effect during normal longitudinal growth. The Igf1 null mouse growth plate demonstrated significant enlargement of the germinal zone; chondrocyte proliferation and numbers were normal but chondrocyte hypertrophy was significantly reduced. In contrast, the Ghr null mouse germinal zone was hypoplastic, chondrocyte proliferation and numbers were significantly reduced, and chondrocyte hypertrophy was also reduced. We have previously demonstrated that IGF-II is highly expressed in growth plate germinal and proliferative zones, so we considered the possibility that GH-stimulated IGF-II production might promote germinal zone expansion and maintain normal proliferation in the Igf1 null mouse growth plate. Supporting this view, IGF-II mRNA was increased in the Igf1 null mouse and decreased in the Ghr null mouse growth plate.Thus, in the complete absence of IGF-I but in the presence of elevated GH in the Igf1 null mouse, reduction in chondrocyte hypertrophy appears to be the major defect in longitudinal bone growth. In the complete absence of a GH effect in the Ghr null mouse, however, both chondrocyte generation and hypertrophy are compromised, leading to a compound deficit in long bone growth. These observations support dual roles for GH in promoting longitudinal bone growth: an IGF-I-independent role in growth plate chondrocyte generation and an IGF-I-dependent role in promoting chondrocyte hypertrophy. The question of whether GH has direct effects on chondrocyte generation is still not settled, however, since it now appears that IGF-II may medicate some of these effects on the growth plate.     

Secretion of insulin-like growth factor II (IGF-II) and IGF-binding protein-2 by intestinal epithelial (IEC-6) cells: implications for autocrine growth regulation.

To identify the factors regulating the proliferation of intestinal epithelium, we examined the effects of various growth factors on [3H] thymidine incorporation into the DNA of IEC-6 cells, an intestinal epithelial cell line derived from rat jejunal crypts. Insulin-like growth factor-I (IGF-I), IGF-II, and insulin stimulated the DNA and protein synthesis of IEC-6 cells in serum-free medium supplemented with transferrin, dexamethasone, and BSA (basal medium). Concentration-response experiments demonstrated that IGF-I is approximately 10 times more potent than IGF-II or insulin in producing 2- to 3-fold stimulations of DNA and protein synthesis by IEC-6 cells. In addition, IEC-6 cells proliferated slowly in the basal medium without any added growth factors. Analysis of medium conditioned by IEC-6 cells by gel filtration chromatography, RIA, HPLC, and N-terminal sequencing revealed that IEC-6 cells synthesize and secrete mature, 7,500 mo wt (M(r)) IGF-II as well as high M(r) forms of IGF-II. In addition, ligand blot, immunoblot, and N-terminal sequence analyses showed that IEC-6 cells produce the 34,000 M(r) IGF-binding protein-2 (IGFBP-2). To determine if IGFBP-2 modulates IGF responses in IEC-6 cells, the IGF-I analogs, Des-(1-3)-IGF-I and [Gln3,Ala4,Tyr15,Leu16]IGF-I, both of which have a reduced affinity for IGFBPs, were tested for their effects on IEC-6 cell proliferation. Both analogs exhibited 10-fold greater potency than IGF-I, presumably because endogenously secreted IGFBPs depress IGF-I binding to cell surface receptors. Finally, purified IGFBP-2 attenuated the DNA synthesis of IEC-6 cells in a dose-dependent manner. We conclude that IGFBP-2 secreted by intestinal epithelial cells is capable of limiting the mitogenic activity of both exogenous and endogenous IGFs by blocking the association of the growth factors with cell surface binding sites. These results further suggest that the growth of IEC-6 cells is modulated by autocrine mechanisms involving IGF-II and IGFBP-2.     

Intact insulin and insulin-like growth factor-I receptor signaling is required for growth hormone effects on skeletal muscle growth and function in vivo

GH and IGF-I are potent regulators of muscle growth and function. Although IGF-I is known to mediate many of the effects of GH, it is not yet clear whether all effects of GH are completely dependent on the IGF-I system. To evaluate the biological effects of the GH/IGF-I axis on muscle growth, we administrated recombinant human GH to mice, which lack IGF-I function specifically in skeletal muscle, due to the overexpression of a dominant-negative IGF-I receptor in this tissue (MKR mice). GH treatment significantly increased the levels of hepatic IGF-I mRNA and serum IGF-I levels in both wild-type (WT) and MKR mice. These GH-induced effects were paralleled by increases in body weight and in the weights of most GH-responsive organs in both groups of mice. Interestingly, unlike WT mice, GH treatment had no effect on skeletal muscle weight in MKR mice. GH treatment failed to reverse the impaired muscle function in MKR mice. Furthermore, MKR mice exhibited no effects of GH on the cross-sectional area of myofibers and the proliferation of satellite cells. Taken together, these data suggest that the in vivo effects of GH on muscle mass and strength are primarily mediated by activation of the IGF-I receptor.     

Enhanced survival and mucosal repair after dextran sodium sulfate-induced colitis in transgenic mice that overexpress growth hormone

BACKGROUND & AIMS: Growth hormone (GH) is used as therapy for inflammatory bowel disease (IBD), but the specific effects on intestine are unknown. Transgenic mice overexpressing GH (MT1-bGH-TG) were used to test whether increased plasma GH levels alter inflammation or crypt damage during dextran sodium sulfate (DSS)-induced colitis. METHODS: MT1-bGH-TG and wild-type (WT) littermates were given 3% DSS for 5 days followed by up to 10 days of recovery. Colitis and epithelial cell proliferation were evaluated histologically. Plasma insulin-like growth factor (IGF)-I and colonic IGF-I, interleukin (IL)-1beta, and intestinal trefoil factor (ITF) messenger RNAs (mRNAs) were measured. RESULTS: DSS induced similar disease onset in MT1-bGH-TG and WT. More MT1-bGH-TG survived than WT. By recovery day 7, MT1-bGH-TG had less inflammation and crypt damage, elevated plasma IGF-I, and increased colonic ITF expression relative to WT. Colonic IL-1beta was elevated in DSS-treated MT1-bGH-TG and WT, but IL-1beta mRNA abundance correlated with disease only in WT. MT1-bGH-TG showed earlier increases in epithelial cell proliferation than WT during recovery but only WT showed atypical repair. CONCLUSIONS: GH does not alter susceptibility to acute DSS-induced colitis but enhances survival, remission of inflammation, and mucosal repair during recovery. GH therapy may be beneficial during active IBD by improving mucosal repair.     

Vascular endothelial growth factor (VEGF164) ameliorates intestinal epithelial injury in vitro in IEC-18 and Caco-2 monolayers via induction of TGF-beta release from epithelial cells

OBJECTIVE: VEGF is a glycoprotein with various (e.g. angiogenic) activities. So far, research has focused on its angiogenic properties. VEGF receptors are localized on epithelial cells of patients with inflammatory bowel disease (IBD) and also on Caco-2 and IEC-18 cells. Our aim was to evaluate the role of VEGF on intestinal epithelial cell (IEC) migration and proliferation by utilizing an established in vitro model. METHODS: IEC-18 and Caco-2 monolayers were wounded with a razor blade as described previously. Cells were incubated in medium w/o rat VEGF(164). After 24 h, migration was assessed by counting cells across the wound edge. Migration was blocked with neutralizing TGF-beta(1) antibodies. IEC proliferation was assessed using the MTT (3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide) test. Semi-quantitative changes of the TGF-beta(1) mRNA expression were evaluated before and after stimulation of the cells with VEGF(164) by RT-PCR. Statistical analysis was performed with ANOVA and the Wilcoxon test. RESULTS: VEGF(164) significantly induced epithelial cell migration in Caco-2 and IEC-18 cells compared to control. TGF-beta(1) antibodies completely abolished this VEGF-induced cell migration. TGF-beta(1) mRNA significantly increased in IEC-18 and Caco-2 cells after stimulation with VEGF. VEGF significantly inhibited epithelial cell proliferation in IEC-18 and in Caco-2 cells, indicating that the observed effects on cell migration were not due to any proliferate effects. CONCLUSION: VEGF effects on epithelial cell migration play an important part in epithelial cell restitution by maintaining mucosal homeostasis after mucosal injury. This effect is mediated by TGF-beta(1). Our results obtain another possible role for increased VEGF levels in the intestinal mucosa of patients with IBD as reported recently by others.     

Spatial distribution of growth hormone receptor, insulin-like growth factor-I receptor and apoptotic chondrocytes during growth plate development

Linear bone growth depends upon proliferation, maturation, and apoptosis of growth plate chondrocytes, processes regulated by growth hormone (GH) and insulin-like growth factor-I (IGF-I). To investigate the contribution of GH, IGF-I and apoptosis to growth plate function, the expression of GH receptor (GHR) and IGF-I receptor (IGF-IR) mRNA were evaluated by in situ hybridization in fractionated costochondral growth plates of growing rats (at 2, 4, and 7 weeks). Apoptosis was determined by TUNEL assay and morphology in histological sections. GHR mRNA was greatest in resting cells with hypertropic cells increasing GHR expression with increasing age. Hypertropic and resting cell IGF-IR mRNA declined over the ages studied. Receptor mRNA expression was altered by exposing cells to GH or IGF-I. GH and IGF significantly decreased GHR mRNA in proliferative cells. GH and IGF also decreased IGF-IR mRNA in resting cells and the 2- and 4-week-old proliferative and hypertropic cells. Treating cells in culture with GH increased the number of apoptotic cells across all ages and zones. Histologically, apoptotic cells were observed at the chondro-osseous junction and within actively proliferating chondrocytes but not in resting cells. Apoptosis was highest at 4 weeks of age with lateral regions displaying the greatest number of cells undergoing apoptosis. These data indicate that apoptosis plays a role in growth plate function, particularly spatial configuration as indicated by the preferential lateral cell apoptosis. The susceptibility of proliferative cells to GHR and IGF-IR down regulation during the period of greatest apoptosis supports a role for the GH-IGF axis in both proliferation and apoptosis during growth plate development.     

Growth hormone reduces the severity of fibrosis associated with chronic intestinal inflammation

BACKGROUND & AIMS: Growth hormone (GH) is used to treat growth delay in children with Crohn's disease and in patients with short-bowel syndrome. GH can increase collagen accumulation in intestinal mesenchymal cells, raising concern that GH therapy could exacerbate fibrosis in patients with Crohn's disease. We tested if GH treatment altered inflammation or fibrosis during chronic, experimental granulomatous enterocolitis. METHODS: Ileum and cecum of Lewis rats were subserosally injected with peptidoglycan-polysaccharide (PG-APS) or control human serum albumin. At the onset of chronic PG-APS-induced inflammation, rats were administered recombinant human GH or vehicle for 14 days. Fibrosis and inflammation were quantified by gross gut disease scoring, histologic scoring, type I collagen, and cytokine expression in cecum. Abundance and localization of suppressor of cytokine signaling-3 (SOCS-3) messenger RNA and/or protein were determined in cecum. Effect of GH, cytokines, or PG-APS on SOCS-3 synthesis was measured in intestinal myofibroblasts. Myofibroblasts overexpressing SOCS-3 were used to test whether SOCS-3 inhibits collagen accumulation. RESULTS: In PG-APS-injected rats, GH modestly reduced gross adhesions and mesenteric contractions, cecal fibrosis score, and collagen expression, but had no effect on intestinal inflammation. GH increased SOCS-3 messenger RNA and protein abundance in PG-APS rats and SOCS-3 messenger RNA was localized to the periphery of granulomas. GH in combination with cytokines or PG-APS, but not alone, induced SOCS-3 synthesis in intestinal myofibroblasts. Myofibroblasts overexpressing SOCS-3 showed reduced cytokine-induced collagen accumulation. CONCLUSIONS: GH modestly reduces intestinal fibrosis associated with chronic experimental enterocolitis and stimulates expression of antifibrogenic SOCS-3, suggesting that GH therapy in inflammatory bowel disease should not exacerbate fibrosis.     

Vascular endothelial growth factor (VEGF164) ameliorates intestinal epithelial injury in vitro in IEC-18 and Caco-2 monolayers via induction of TGF-β release from epithelial cells

Objective. VEGF is a glycoprotein with various (e.g. angiogenic) activities. So far, research has focused on its angiogenic properties. VEGF receptors are localized on epithelial cells of patients with inflammatory bowel disease (IBD) and also on Caco-2 and IEC-18 cells. Our aim was to evaluate the role of VEGF on intestinal epithelial cell (IEC) migration and proliferation by utilizing an established in vitro model. Methods. IEC-18 and Caco-2 monolayers were wounded with a razor blade as described previously. Cells were incubated in medium w/o rat VEGF₁₆₄. After 24?h, migration was assessed by counting cells across the wound edge. Migration was blocked with neutralizing TGF-β₁ antibodies. IEC proliferation was assessed using the MTT (3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide) test. Semi-quantitative changes of the TGF-β₁ mRNA expression were evaluated before and after stimulation of the cells with VEGF₁₆₄ by RT-PCR. Statistical analysis was performed with ANOVA and the Wilcoxon test. Results. VEGF₁₆₄ significantly induced epithelial cell migration in Caco-2 and IEC-18 cells compared to control. TGF-β₁ antibodies completely abolished this VEGF-induced cell migration. TGF-β₁ mRNA significantly increased in IEC-18 and Caco-2 cells after stimulation with VEGF. VEGF significantly inhibited epithelial cell proliferation in IEC-18 and in Caco-2 cells, indicating that the observed effects on cell migration were not due to any proliferate effects. Conclusion. VEGF effects on epithelial cell migration play an important part in epithelial cell restitution by maintaining mucosal homeostasis after mucosal injury. This effect is mediated by TGF-β₁. Our results obtain another possible role for increased VEGF levels in the intestinal mucosa of patients with IBD as reported recently by others.     

Glucagon-like peptide-2 activates beta-catenin signaling in the mouse intestinal crypt: role of insulin-like growth factor-I

Chronic administration of glucagon-like peptide-2 (GLP-2) induces intestinal growth and crypt cell proliferation through an indirect mechanism requiring IGF-I. However, the intracellular pathways through which IGF-I mediates GLP-2-induced epithelial tropic signaling remain undefined. Because beta-catenin and Akt are important regulators of crypt cell proliferation, we hypothesized that GLP-2 activates these signaling pathways through an IGF-I-dependent mechanism. In this study, fasted mice were administered Gly(2)-GLP-2 or LR(3)-IGF-I (positive control) for 0.5-4 h. Nuclear translocation of beta-catenin in non-Paneth crypt cells was assessed by immunohistochemistry and expression of its downstream proliferative markers, c-myc and Sox9, by quantitative RT-PCR. Akt phosphorylation and activation of its targets, glycogen synthase kinase-3beta and caspase-3, were determined by Western blot. IGF-I receptor (IGF-IR) and IGF-I signaling were blocked by preadministration of NVP-AEW541 and through the use of IGF-I knockout mice, respectively. We found that GLP-2 increased beta-catenin nuclear translocation in non-Paneth crypt cells by 72 +/- 17% (P < 0.05) and increased mucosal c-myc and Sox9 mRNA expression by 90 +/- 20 and 376 +/- 170%, respectively (P < 0.05-0.01), with similar results observed with IGF-I. This effect of GLP-2 was prevented by blocking the IGF-IR as well as ablation of IGF-I signaling. GLP-2 also produced a time- and dose-dependent activation of Akt in the intestinal mucosa (P < 0.01), most notably in the epithelium. This action was reduced by IGF-IR inhibition but not IGF-I knockout. We concluded that acute administration of GLP-2 activates beta-catenin and proliferative signaling in non-Paneth murine intestinal crypt cells as well as Akt signaling in the mucosa. However, IGF-I is required only for the GLP-2-induced alterations in beta-catenin.     

Investigation of insulin-like growth factor (IGF)-I and insulin receptor binding and expression in jejunum of parenterally fed rats treated with IGF-I or growth hormone.

To investigate the ability of insulin-like growth factor-I (IGF-I), but not GH, to stimulate jejunal growth, we compared indices of IGF-I and insulin receptor expression in jejunal membranes from rats maintained with total parenteral nutrition (TPN) and treated with rhIGF-I and/or rhGH. TPN without growth factor treatment (TPN control) induced jejunal atrophy, reduced serum IGF-I, increased serum insulin concentrations, and increased IGF-I receptor number, IGF-I receptor messenger RNA, and insulin-specific binding to 133% to 170% of the orally fed reference values, P < 0.01. Compared with TPN control, IGF-I or IGF-I + GH stimulated jejunal mucosal hyperplasia; IGF-I treatment increased serum IGF-I by 2- to 3-fold and decreased serum insulin concentrations by 60%, decreased IGF-I receptor number by 50% (P < 0.001), and increased insulin receptor affinity and insulin receptor protein content. Treatment with GH alone increased serum IGF-I concentration, did not alter TPN-induced jejunal atrophy, and decreased insulin-specific binding and insulin receptor protein content (39% and 59%, respectively, of the TPN control values, P < 0.01). We conclude that: 1) jejunal IGF-I receptor content reflects circulating concentration of ligand and is not limiting for jejunal growth; and 2) increased circulating concentration of IGF-I may promote jejunal growth via interaction with jejunal insulin or IGF-I receptors.     

Insulin resistance-inducing cytokines differentially regulate SOCS mRNA expression via growth factor- and Jak/Stat-signaling pathways in 3T3-L1 adipocytes

Various cytokines, including tumor necrosis factor (TNF) alpha, growth hormone (GH) and interleukin (IL)-6, induce insulin resistance. Recently, it was demonstrated that induction of suppressor of cytokine signaling (SOCS)-3 by TNFalpha and GH is an important mechanism by which these cytokines impair insulin sensitivity. The current study investigated in 3T3-L1 adipocytes whether TNFalpha and GH also upregulate SOCS-1 and SOCS-6, which have both been shown to inhibit insulin signaling potently, and whether IL-6 might alter synthesis of SOCS-1, -3 and -6. Interestingly, 10 ng/ml TNFalpha, 500 ng/ml GH and 30 ng/ml IL-6 induced SOCS-1 mRNA time-dependently with maximal stimulation detectable after 8 h of TNFalpha and 1 h of GH and IL-6 addition respectively. Furthermore, TNFalpha and GH caused sustained upregulation of SOCS-1 for up to 24 h, whereas stimulation by IL-6 was only transient, with SOCS-1 mRNA returning to basal levels 2 h after effector addition. Induction of SOCS-1 was dose-dependent, and significant stimulation was detectable at concentrations as low as 3 ng/ml TNFalpha, 50 ng/ml GH and 10 ng/ml IL-6. Furthermore, stimulation experiments and studies using pharmacologic inhibitors suggested that the positive effect of TNFalpha, GH and IL-6 on SOCS-1 mRNA is, at least in part, mediated by Janus kinase (Jak) 2. Finally, SOCS-3 expression was dose- and time-dependently induced by IL-6, at least in part via Jak2, but none of the cytokines affected SOCS-6 expression. Taken together, our results show a differential regulation of SOCS mRNA by insulin resistance-inducing hormones, and suggest that SOCS-1, as well as SOCS-3, may be an important intracellular mediator of insulin resistance in fat cells and a potential pharmacologic target for the treatment of impaired insulin sensitivity.     

Effect of growth hormone and insulin-like growth factor I (IGF-I) on the expression of IGF-I messenger ribonucleic acid and peptide in rat tibial growth plate and articular chondrocytes in vivo

Conflicting data exist as to whether insulin-like growth factor I (IGF-I) messenger RNA (mRNA) and peptide are expressed within chondrocytes. This question is pertinent to the mode of GH action on longitudinal bone growth. We have, therefore, investigated this issue in normal rats and in hypophysectomized rats treated for 24 h with GH or IGF-I using in situ hybridization and immunohistochemistry. Serum IGF-I, body weight, and tibial growth plate, but not articular cartilage, height increased with both treatments. Both IGF-I mRNA and IGF-I immunoreactivity occurred in all chondrocyte layers of growth plate and articular cartilage. The percentage of cells with IGF-I mRNA correlated well with IGF-I immunoreactivity under all experimental conditions. In normal rats, IGF-I expression was highest in the upper hypertrophic zone in growth plate (68-71%) and articular cartilage (32-34%). Hypophysectomy, GH, or IGF-I did not significantly affect this percentage. In the stem cell and proliferative and lower hypertrophic zones of growth plate, hypophysectomy dramatically reduced the percentage of labeled chondrocytes, and GH restored it. IGF-I increased IGF-I mRNA and immunoreactivity only in the proliferative zone. In articular cartilage, both remained unchanged under all experimental conditions. Together with our previous finding that GH infusion of hypophysectomized rats enhances chondrocyte maturation at all differentiation stages, the present results are compatible with the idea that IGF-I produced by all chondrocyte layers under the influence of GH mediates chondrocyte maturation and thus longitudinal bone growth in an autocrine/paracrine manner.