Skeletal unloading induces resistance to insulin-like growth factor I on bone formation

Skeletal unloading results in an inhibition of bone formation associated with a decrease in osteoblast number, impaired mineralization of bone, and altered proliferation and differentiation of osteoprogenitor cells. Although such changes are likely to be mediated by multiple factors, resistance to the growth-promoting action of insulin-like growth factor I (IGF-I) has been hypothesized to play an important role. To determine whether skeletal unloading induces resistance to IGF-I on bone formation, we examined the response of unloaded (hindlimb elevation) and normally loaded tibia and femur to IGF-I administration. To eliminate the variable of endogenous growth hormone production and secretion during exogenous IGF-I administration, we used growth hormone-deficient dwarf rats (dw-4). The rats were given IGF-I (2.5 mg/kg/day) or vehicle during 7 and 14 days of unloading or normal loading. This significantly increased the serum level of IGF-I in both the normally loaded and unloaded rats. Unloading did not affect the serum level of IGF-I in the vehicle-treated rats. IGF-I markedly increased periosteal bone formation at the tibiofibular junction of normally loaded rats. Unloading decreased bone formation in the vehicle-treated rats, and blocked the ability of IGF-I to increase bone formation. On the other hand, IGF-I increased periosteal bone formation at the midpoint of the humerus (normally loaded in this model) in both hindlimb-elevated and normally loaded rats. IGF-I significantly increased osteogenic colony number, total ALP activity, and total mineralization in bone marrow osteoprogenitor (BMOp) cells of normally loaded rats. Unloading reduced these parameters in the vehicle-treated rats, and blocked the stimulation by IGF-I. Furthermore, IGF-I administration (10 ng/ml) in vitro significantly increased cell proliferation of the BMOp cells isolated from normally loaded bone, but not that of cells from unloaded bone. These results indicate that skeletal unloading induces resistance to IGF-I on bone formation.     

Aging impairs IGF-I receptor activation and induces skeletal resistance to IGF-I.

IGF-I plays an important anabolic role in stimulating bone formation and maintaining bone mass. We show that the pro-proliferative, anti-apoptotic, and functional responses to IGF-I in bone and BMSCs decrease with aging. These changes are associated with impaired receptor activation and signal transduction through the MAPK and PI3K pathways. INTRODUCTION: IGF-I is a potent anabolic agent having effects across diverse tissues and cell types. With aging, bone becomes resistant to the anabolic actions of IGF-I. To examine the effects of aging on bone responsiveness to IGF-I, we measured the pro-proliferative, anti-apoptotic, and functional responses of bone and bone marrow stromal cells (BMSCs) to IGF-I and evaluated IGF-I signal transduction in young, adult, and old mice. MATERIALS AND METHODS: Male C57BL/6 mice 6 wk (young), 6 mo (adult), and 24 mo (old) were treated with IGF-I for 2 wk using osmotic minipumps, and osteoblast proliferation (BrdU labeling) in vivo, and osteoprogenitor number (BMSC culture and calcium nodule formation) were measured. Proliferation, apoptosis, and expression of key osteoblast factors (alkaline phosphatase, collagen, osteocalcin, RANKL, osteoprotegerin (OPG), macrophage-colony stimulating factor [M-CSF]) and IGF-I signaling elements and their activation in IGF-I-treated cells were studied using QRT-PCR and Western blot analysis. Data were analyzed using ANOVA. RESULTS: Aging decreased the basal and IGF-I-stimulated number of BrdU-labeled osteoblasts and reduced the ability of IGF-I to stimulate osteoprogenitor formation (calcium nodule number) by 50%. The pro-proliferative and anti-apoptotic actions of IGF-I were blunted in cells from old animals. These changes were accompanied by age-related alterations in the ability of IGF-I to regulate alkaline phosphatase, collagen, osteocalcin, RANKL, OPG, and M-CSF expression. IGF-I binding was normal, but IGF-I receptor mRNA and protein expression was increased in aged animals by 2- and 10-fold, respectively. The age-related changes in proliferation, apoptosis, and function were accompanied by loss of IGF-I-induced signaling at the receptor level and at key regulatory sites along the MAPK (ERK1/2) and PI3K (AKT) pathways. CONCLUSIONS: Our data show that aging is accompanied by loss of bone and BMSC/osteoblast responsiveness to IGF-I and that these changes are associated with resistance to IGF-I signaling that involve receptor activation and downstream signaling events.     

Enhanced reinnervation of the paralyzed orbicularis oculi muscle after insulin-like growth factor-I (IGF-I) delivery to a nerve graft

Facial paralysis (FP) remains today one of the most disturbing cranial nerve disorders. The present study utilized the rat model of FP and examined a dual approach of combining the current microsurgical treatment of cross-facial nerve graft (CFNG) with local administration of insulin-like growth factor-I (IGF-I). The efficacy of this combined treatment approach was assessed by motor end-plate analysis of the reinnervated orbicularis oculi muscle (OOM). Local administration of IGF-I (50 microg/ml) to the CFNG demonstrated a 61 percent increase in the number of end-plates in the reinnervated OOMs, compared to the OOMs reinnervated with CFNG plus vehicle. These results indicate that the local therapeutic augmentation of IGF-I levels at the coaptation site(s) of the CFNG may, in fact, enhance reinnervation of muscle and recovery of function in general.     

In vivo actions of insulin-like growth factor-I (IGF-I) on bone formation and resorption in rats

The in vivo action of insulin-like growth factor-I on bone metabolism has been studied using a new model. Insulin-like growth factor-I (IGF-I) was continuously infused into the arterial supply of the right hindlimb of ambulatory rats for up to 14 days and the effects on cortical and trabecular bone formation and the number of osteoclasts were determined by histomorphometric techniques. The contralateral limb acted as an internal control. IGF-I infusion significantly increased cortical bone formation (p less than 0.01). Trabecular bone was increased 22% (p = 0.07), but the infusion was only for seven days. These effects of IGF-I were age dependent, being absent in young, rapidly growing animals, but present at least until one year of age. IGF-I appears to be a purely anabolic hormone for bone formation, since it significantly stimulates osteoblasts and decreases the number of osteoclasts. Thus, although IGF-I mediates the growth-promoting effect of growth hormone, it does not mediate growth hormone's action on bone resorption.     

胰岛素样生长因子1对结肠癌细胞凋亡的影响及其机制研究

目的研究胰岛素样生长因子1(IGF-I)通过磷酯酰肌醇3-激酶/蛋白激酶B(PI-3K/Akt)信号通路对结肠癌细胞株SW480凋亡率的影响及凋亡抑制蛋白survivin表达水平的变化。方法在培养结肠癌SW480细胞株时,采用不同浓度的IGF-I不同时间作用于SW480细胞,然后采用Western blot检测凋亡抑制蛋白survivin表达变化。100 ng/mL IGF-I作用于SW480细胞2 h内,Westernblot检测Akt的磷酸化表达变化;Akt特异性抑制剂LY294002处理前后,利用Western blot检测Akt磷酸化表达的变化,Western blot及细胞免疫荧光检测survivin表达的变化,流式细胞术检测细胞凋亡率的改变。结果IGF-I能上调SW480细胞的survivin表达,且其表达水平与IGF-I的作用时间、浓度呈依赖性(IGF-I作用SW480细胞不同时间后,survivin蛋白表达显著升高,并在12 h时达到峰值,所有实验组survivin蛋白的表达均显著高于对照组;IGF-I浓度为100 ng/mL时,survivin蛋白的表达达到顶峰);IGF-I快速激活SW480细胞中PI-3K/Akt信号通路(IGF-I作用SW480细胞15,30 min后,p-Akt蛋白的表达达到高峰);IGF-I能通过PI-3K/Akt信号通路上调SW480细胞survivin的表达;IGF-I能通过PI-3K/Akt信号通路抑制SW480细胞凋亡[利用流式细胞术检测空白组、刺激组、阻断组各组SW480细胞的凋亡率,分别为(5.18±0.415)%,(0.85±0.052)%,(3.15±0.411)%,各组之间差异有统计学意义(P0.05)]。结论IGF-I可通过PI-3K/Akt通路诱导survivin表达,从而抑制结肠癌细胞SW480的凋亡。     

Gene expression of insulin-like growth factor-I and IGF-I receptor during wound healing in rats.

OBJECTIVE--To measure the time course of changes in the concentrations of insulin-like growth factor-I (IGF-I), IGF-I messenger RNA (mRNA), and IGF-I receptor mRNA during wound healing. DESIGN--Open experimental study. MATERIAL--40 male Sprague-Dawley rats, each weighing 300 g. INTERVENTION--Stainless steel wire mesh cylinders implanted in the subcutaneous tissue of the back. Five rats killed at each time point (1.5, 2, 3, 4, 5, 7, 11, and 18 weeks). MAIN OUTCOME MEASURES--Wet weight and concentrations of IGF-I mRNA, and IGF-I receptor mRNA of granulation tissue, and concentration of IGF-I in wound fluid. RESULTS--The wet weight of granulation tissue increased significantly between week 1.5 and weeks 4-5, and then decreased. IGF-I mRNA concentration (amol/microgram DNA) increased significantly (threefold) between week 1.5 and weeks 3-5, and decreased between weeks 5 and 7. The concentration of IGF-I receptor RNA remained constant throughout the study, and the concentration of IGF-I in wound fluid remained constant until week 8 and then increased to a higher level at weeks 11-18. CONCLUSION--There is a transient rise in the gene expression of IGF-I but not of IGF-I receptor mRNA during wound healing.     

Gene mediated insulin-like growth factor-I delivery to the synovium.

The feasibility of articular gene therapy using insulin-like growth factor-I transgene expression in synovial tissues was assessed in vitro by transfection of synovial explant and monolayer cultures. Synovial membrane was harvested from horses and distributed for explant culture in multiwell plates or digested for monolayer culture in multiwell plates and chamber slides. Synovial monolayers were cultured for 48 h after infection with 0, 100, 200, or 500 moi adenovirus-IGF-I (AdeIGF-I) to establish an optimum dose. Explants were then either infected with AdeIGF-I or adenoviral LacZ and cultured for 8 days, treated with 100 ng/ml recombinant IGF-I as a positive control, or remained as uninfected untreated culture controls. Expression of IGF-I in explants and monolayers was assessed by in situ hybridization and quantitative polymerase chain reaction (PCR), and translation confirmed by IGF-I radioimmunoassay (RIA) and tissue immunoreaction. Effects of IGF-I on synovial function was assessed by proteoglycan and hyaluronan assay, and northern blot assessment of decorin and collagen type I expression. Significant transgene expression in synovial cells was present for all AdeIGF-I concentrations. Similarly, medium IGF-I concentrations were significantly elevated in AdeIGF-I infected synovial monolayer and explant cultures at all time points. Peak IGF-I concentration of 246 +/- 43 ng/ml developed in explant cultures on day 4; IGF-I levels in control explant groups were unchanged over baseline values. In situ hybridization and immunolocalization for IGF-I indicated focal IGF-I expression in intimal and subintimal layers of infected explants, with diffuse immunoreaction throughout infected subintimal and fibrous layers. For monolayer cultures, intracellular immunoreaction to IGF-I was markedly higher in infected cells, and was most prominent at 100 moi. Effects of IGF-I on synoviocyte cultures were evident on northern blots, which showed decreased decorin expression and elevated type I collagen production in AdeIGF-I infected monolayers. Proteoglycan concentration in the medium from explant cultures rose over the initial 4 days but was similar between treatment groups. The concentration of hyaluronan in medium from explant cultures did not differ significantly within or between treated and control groups during the 8-day study period. These data indicate that IGF-I can be successfully introduced to synovial structures by adenoviral vectors and results in effective IGF-I ligand synthesis without untoward synovial morphologic effects.     

Altered postnatal expression of insulin-like growth factor-I (IGF-I) and type X collagen preceding the Perthes' disease-like lesion of a rat model.

The spontaneously hypertensive rat (SHR) is a widely used animal model for the study of hypertension. It also exhibits an osteonecrosis of the femoral epiphysis that resembles the clinical features of Perthes' disease in humans. In this rat model, occlusion of the epiphyseal vessels occurs as a result of a breakdown of the mechanically vulnerable epiphysis. The postnatal development of the epiphysis recapitulates the serial events of the endochondral ossification (i.e., cartilage formation), chondrocyte hypertrophy, cartilage mineralization, vascularization, and introduction of osteoblasts that form the secondary ossification center within the epiphysis. In the present study, a detailed radiographic and histological analysis demonstrates that the osteonecrosis is preceded by a disturbance of the cartilage mineralization and a disturbance of the ossification, despite a normal hypertrophy of the epiphyseal cartilage. These observations suggest that abnormal development of the femoral epiphysis occurs much earlier than manifestation of the osteonecrosis. They lead us to a hypothesis that yet-unclarified transitional events between the cartilage hypertrophy and the cartilage mineralization may be affected in SHRs. Type X collagen is a developmentally regulated matrix molecule that is implicated in the mineralization of the hypertrophied chondrocytes. We show that the expression of type X collagen during epiphyseal ossification is delayed in SHRs (vs. normal controls), suggesting disturbed growth and/or differentiation of the epiphyseal chondrocytes. Postnatal growth and differentiation of the chondrocytes at least partly depend on insulin-like growth factor-I (IGF-I), which is produced by the chondrocytes in response to the pituitary growth hormone and stimulates cartilage growth in situ. The present study demonstrates an altered IGF-I expression during early postnatal life in SHRs and suggests that the altered IGF-I expression as well as the following delay in upregulation of type X collagen may cause the mechanical vulnerability of the femoral epiphysis in SHRs.     

In vivo and in vitro effects of insulin-like growth factor-I (IGF-I) on femoral mRNA expression in old rats

The in vivo response of bone to IGF-I infusion in a marrow ablation model and the effect of IGF-I on bone marrow stromal cells in vitro was evaluated. IGF-I (25 ng/day), infused directly into femur, stimulated the expression of alkaline phosphatase, procollagen ∝₁ (I) and osteopontin mRNA, while osteocalcin mRNA was not affected. The dose dependency to IGF-I was bi-phasic, with stimulation at 25 and 50 ng but not at 150 ng/day. The effect of IGF-I was observed in the aged but not in the adult rat femur. However, the elevated mRNA levels in old bones with IGF-I treatment were still below those observed in adult bones. The effect of IGF-I was also examined in cultured stromal cells. IGF-I (50 ng/ml) stimulates the expression of alkaline phosphatase, procollagen ∝₁ (I), osteopontin and osteocalcin mRNA in stromal cells from both adult and old rats. These results suggest that the lack of response of adult bone to IGF-I in vivo was not due to the impaired response of the stromal cells to IGF-I. Differences in the responses of stromal cells from adult and old animals were noted. In the presence of serum (10%), stromal cells from adult rats were stimulated to synthesize DNA at lower levels of IGF-I than stromal cells from old animals. Our results show that IGF-I can stimulate mRNA expression of osteoblast markers in vivo in aged rats in a marrow ablation model and enhance DNA synthesis and gene expression in cultured marrow stromal cells from old rats. Thus, it is possible that exogenous IGF-I could be beneficial in treating age-associated osteopenia.     

The beneficial effects of recombinant human insulin-like growth factor-I (IGF-I) on wound healing in severely wounded senescent mice

The effects of recombinant insulin-like growth factor I (rIGF-I) on wound healing were tested using senescent and young BDF-1 mice, aged 108 weeks and 10 weeks, respectively. After inflicting a full thickness dermal burn encompassing 15% of the body surface, a skin incision, 2 cm in length, was made in the back. A silicone tube containing a piece of polyvinyl sponge was then implanted into a subcutaneous pocket in the flank to collect body fluid. An osmotic pump was buried in the abdominal subcutaneous tissue for the continuous infusion of rIGF-I, the control being treated with the solvent of IGF-I, physiological saline, only. The administration of IGF-I produced favorable effects on wound healing in the senescent mice, shown by enhanced tensile strength and an elevated concentration in the hydroxyproline of the polyvinyl sponge content. The IGF-I-treated severely wounded senescent mice healed, better than their counterparts and their skeletal muscles contained more glutamine. Furthermore, they showed more enhanced cutaneous hypersensitivity towards dinitrofluorobenzene than the controls, suggesting an enhanced grade of cellular immunity. There were no conspicuous differences between the two groups of young mice. These data may suggest the beneficial effects of rIGF-I on wound healing, especially in geriatric surgery.     

Relationships between insulin-like growth factor-I (IGF-I) and OPG, RANKL, bone mineral density in healthy Chinese women

Summary Serum IGF-I level was negatively correlated with OPG and OPG/RANKL ratio, but positively correlated with RANKL. Serum OPG level in the highest quintile of IGF-I was significantly lower than that in the lowest. We conclude that the effect of IGF-I on bone remodeling may be mediated by the OPG/RANKL system. Introduction Insulin-like growth factor I (IGF-I) is an important factor in coupling bone remodeling, activating both formation and resorption. Compared with the many studies on the role of IGF-I in bone formation, the information regarding its effects on bone resorption is limited and conflicting. The balance of the two peptides produced by osteoblasts, osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL), is critical for the bone resorption process. Our study was designed to analyze the relationships of serum concentrations of IGF-I with OPG, RANKL, OPG/RANKL ratio as well as BMDs in healthy Chinese women. Methods BMDs at lumbar spine and proximal femur in 504 pre- and postmenopausal women were measured by DXA. Serum levels of IGF-I, OPG and RANKL were also measured. Pearson’s correlation and partial correlation analysis, ANOVA, covariance analysis and stepwise multiple regression analysis were used as appropriate. Results Age was negatively correlated with serum levels of IGF-I (r = −0.702, p < 0.001). IGF-I was negatively correlated with OPG and OPG/RANKL ratio, but positively correlated with RANKL. The relationship between IGF-I and BMDs disappeared after adjustment for age. In postmenopausal women, IGF-I was lower in women with osteoporosis than in those with normal BMD (p = 0.056), but no differences were found among OPG, RANKL and OPG/RANKL ratio. Serum levels of OPG in the highest quintile of IGF-I were significantly lower than those in the lowest quintile of IGF-I, while no difference was found in RANKL. In the multiple regression analysis model, serum levels of IGF-I were the main determinants of the bone mass in Chinese women. Conclusions In conclusion, the relationship between decreasing IGF-I and BMDs in healthy Chinese women influenced by age, whereas the effect of IGF-I on bone remodeling (bone resorption) may be mediated by the OPG/RANKL system.     

Differential effects of insulin and insulin-like growth factor-I in the femoral tissues of rats with skeletal unloading

The alteration of bone metabolism in the femur of rats with skeletal unloading for 4 days was investigated. Skeletal unloading was designed using the model of hindlimb hang in rats. Skeletal unloading caused a significant decrease in femoral weight, calcium, and phosphorus contents in the metaphysis but not diaphysis. Also, the unloading induced a significant decrease of zinc content, alkaline phosphatase activity, and deoxyribonucleic acid (DNA) content in the femoral diaphysis and metaphysis. When the femoraldiaphyseal and metaphyseal tissues from normal and skeletal-unloading rats were cultured in the presence of insulin (10^-9 and 10^-8 M) for 24 hours in vitro, the hormonal effect to increase alkaline phosphatase activity and DNA content in the diaphysis, but not metaphysis, was lost in the bone tissues from unloading rats. However, the culture with insulin-like growth factor-I (IGF-I; 10^-8 and 10^-7 M) produced a significant increase of alkaline phosphatase activity and DNA content in both the diaphyseal and metaphyseal tissues from normal and unloading rats. These results demonstrate that skeletal unloading causes an impairment of insulin effect, but not IGF-I effect, on bone metabolism in femoral tissues.     

Circulating levels of insulin-like growth factor binding protein 3 (IGFBP-3) and insulin-like growth factor I (IGF-I) in perimenopausal women

The study investigated possible menopause-related changes in circulating insulin-like growth factor binding protein 3 (IGFBP-3) levels and their relationship with insulin-like growth factor I (IGF-I) plasma levels. Forty-three healthy women, aged 45–55 years, were studied (22 premenopausal and 21 postmeno-pausal, matched for age and body mass index); in all subjects plasma IGF-I and IGFBP-3 levels were measured by radioimmunoassay. No difference was found between mean IGFBP-3 plasma levels in the two groups studied (premenopausal 3.42±0.49 v postmenopausal 3.46±0.58 mg/l), while mean IGF-I levels were significantly lower in postmenopausal as compared with premenopausal women (136.7±37.86 v 175.7±51.91 ng/ml,p<0.02). Multiple regression analysis showed no significant effect of age, body mass index and years since menopause on IGFBP-3 levels; however, considering the IGF-I/IGFBP-3 ratio as a possible parameter of circulating free somatomedin C, an inverse correlation was found with years since menopause (n=43,r=–0.499,p<0.001). We conclude that lack of oestrogen induces different effects on circulating IGF-I and IGFBP-3, possibly reflecting a real decrease in IGF-I activity.     

Keloid fibroblast responsiveness to epidermal growth factor and activation of downstream intracellular signaling pathways.

Keloids, which overgrow the boundaries of the original injury, represent aberrations in the fundamental process of wound healing that include over-abundant cell in-migration, cell proliferation, and inflammation, as well as increased extracellular matrix synthesis and defective remodeling. To understand the key events that result in the formation of these abnormal scars would open new avenues for better understanding of excessive repair, and might provide new therapeutic options. We examined epidermal growth factor receptor (EGFR)-induced cell motility in keloid fibroblasts, as this receptor initiates cell migration during normal wound repair. We show that keloid fibroblasts respond to EGF-induced cell migration but the response is somewhat diminished compared to normal adult fibroblasts (approximately 30% reduced); the mitogenic response was similarly blunted (approximately 5% reduced). Keloid fibroblasts express near normal levels of EGFR (82%), but show a much more attenuated activation of EGFR itself and the motility-associated phospholipase C-gamma. This was reflected in part by rapid loss of EGFR upon exposure to EGF. Interestingly, while extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) activation was relatively robust in keloid fibroblasts, the downstream triggering of the motility-associated calpain activity was blunted. This was reflected by high cell-substratum adhesiveness in the keloid fibroblasts. Thus, the blunted migratory response to EGF noted in keloid fibroblasts appears due to limited activation of two important biochemical switches for cell motility.     

Skeletal unloading-induced insulin-like growth factor 1 (IGF-1) nonresponsiveness is not shared by platelet-derived growth factor: the selective role of integrins in IGF-1 signaling

Integrin receptors bind extracellular matrix proteins, and this link between the cell membrane and the surrounding matrix may translate skeletal loading to biologic activity in osteoprogenitor cells. The interaction between integrin and growth factor receptors allows for mechanically induced regulation of growth factor signaling. Skeletal unloading leads to decreased bone formation and osteoblast proliferation that can be explained in part by a failure of insulin-like growth factor 1 (IGF-1) to activate its signaling pathways in unloaded bone. The aim of this study is to determine whether unloading-induced resistance is specific for IGF-1 or common to other skeletal growth factors, and to examine the regulatory role of integrins in IGF-1 signaling. Bone marrow osteoprogenitor (BMOp) cells were isolated from control or hindlimb suspended rats. Unloaded BMOp cells treated with IGF-1 failed to respond with increased proliferation, receptor phosphorylation, or signaling activation in the setting of intact ligand binding, whereas the platelet-derived growth factor (PDGF) response was fully intact. Pretreatment of control BMOp cells with an integrin inhibitor, echistatin, failed to disrupt PDGF signaling but blocked IGF-1 signaling. Recovery of IGF-1 signaling in unloaded BMOp cells followed the recovery of marked reduction in integrin expression induced by skeletal unloading. Selective targeting of integrin subunits with siRNA oligonucleotides revealed that integrin β1 and β3 are required for normal IGF-1 receptor phosphorylation. We conclude that integrins, in particular integrin β3, are regulators of IGF-1, but not PDGF, signaling in osteoblasts, suggesting that PDGF could be considered for investigation in prevention and/or treatment of bone loss during immobilization and other forms of skeletal unloading.     

The effect of dynamic compression on the response of articular cartilage to insulin-like growth factor-I.

Articular cartilage is routinely subjected to mechanical forces and to cell-regulatory molecules. Previous studies have shown that mechanical stimuli can influence articular chondrocyte metabolic activity, and biochemical studies have shown that growth factors and cytokines control many of the same cell functions. Little is known, however, of the relationships or interplay, if any, between these two key components of the articular environment. This study investigated the comparative and interactive effects of low amplitude, sinusoidal, dynamic compression and insulin-like growth factor-I (IGF-I), a polypeptide in synovial fluid that is anabolic for cartilage. In bovine patellofemoral cartilage explants, IGF-I increased protein and proteoglycan synthesis 90% and 120%, respectively while dynamic compression increased protein and proteoglycan synthesis 40% and 90%, respectively. Stimulation by IGF-I was significantly greater than by dynamic compression for both protein and proteoglycan synthesis. When applied together, the two stimuli enhanced protein and proteoglycan synthesis by 180% and 290%, respectively, a degree greater than that achieved by either stimulus alone. IGF-I augmented protein synthesis with a time constant of 12.2 h. Dynamic compression increased protein synthesis with a time constant of 2.9 h, a rate significantly faster than that of IGF-I, suggesting that these signals act via distinct cell activation pathways. When used together, dynamic compression and IGF-I acted with a time constant of 5.6 h. Thus, dynamic compression accelerated the biosynthetic response to IGF-I and increased transport of IGF-I into the articular cartilage matrix, suggesting that, in addition to independently stimulating articular chondrocytes, cyclic compression may improve the access of soluble growth factors to these relatively isolated cells.     

Signaling through the small G-protein Cdc42 is involved in insulin-like growth factor-I resistance in aging articular chondrocytes.

During aging, chondrocytes become unresponsive to insulin-like growth factor-I (IGF-I). This study examined the role of Cdc42 (cell-division-cycle 42) in IGF-I signaling during aging. Experiments were performed using cartilage and chondrocytes isolated from horses ages 1 day-25 years. Northern analysis was used to examine expression of the small GTPases Cdc42, Rac, and RhoA. Western analysis was utilized to assess total Cdc42 (GTP + GDP-bound); active, GTP-Cdc42 was assessed using a pulldown assay with Western analysis. GTP-Cdc42 was also measured following IGF-I treatment. Gene expression for Cdc42 and Rac were decreased in mature samples, but there was no difference in total Cdc42 (GTP + GDP-bound) protein expression due to age. GTP-Cdc42 was significantly greater in prepubescent samples compared to other age groups. IGF-I diminished the GTP-bound state of Cdc42 in prepubescent chondrocytes; however, this effect was lost during aging. No differences in results were observed due to sample type; that is, cartilage tissues versus isolated chondrocytes. These studies suggest that loss of IGF-I-mediated regulation of Cdc42 activation may be a mechanism for the chondrocyte unresponsive state during aging. Further, the activation state of Cdc42, measured in native and IGF-I-treated cartilage tissue for the first time, is similar to that of isolated chondrocytes, indicating that the activation state of small G-proteins is not affected by isolation of chondrocytes from the extracellular matrix. Continued studies will identify the upstream regulators of Cdc42, which will further elucidate the molecular mechanism of IGF-I resistance during aging thereby providing insight into targeted strategies for age-related osteoarthritis.     

Continuous infusion of insulin-like growth factor-I into the epiphysis of the tibia

We have developed a method to promote longitudinal bone growth at the level of a specific growth-plate (GP) in young rabbits. Insulin-like growth factor-I (IGF-I) was continuously infused by means of an osmotic pump into the bone marrow cavity of the proximal epiphysis of the tibia. Radiological measurement showed a 2-mm overgrowth of the tibia after 4 weeks of treatment, while histological analysis demonstrated a 15% increase in the thickness of the selected GP. The local infusion of IGF-I increased the numbers of both proliferative and hypertrophic chondrocytes and promoted hyperplasia of bony trabeculae within the epiphysis. The distribution of material infused locally into the epiphysis was simulated by the infusion of Indian ink using the same methodology (osmotic pump) as that for IGF-I. Most of the dye remained within the bone marrow cavity of the epiphysis, but a portion infiltrated into the GP, reaching the deep layer of the physeal chondrocytes and primary spongiosa of the metaphysis. These results suggest that the method reported here is a valid one for delivering cytokines or growth factors to the selected GP and for controlling the growth and differentiation of physeal chondrocytes.