Thyroid hormone‐mediated growth and differentiation of growth plate chondrocytes involves IGF‐1 modulation of β‐catenin signaling

Thyroid hormone regulates terminal differentiation of growth plate chondrocytes in part through modulation of the Wnt/β‐catenin signaling pathway. Insulin‐like growth factor 1 (IGF‐1) has been described as a stabilizer of β‐catenin, and thyroid hormone is a known stimulator of IGF‐1 receptor expression. The purpose of this study was to test the hypothesis that IGF‐1 signaling is involved in the interaction between the thyroid hormone and the Wnt/β‐catenin signaling pathways in regulating growth plate chondrocyte proliferation and differentiation. The results show that IGF‐1 and the IGF‐ receptor (IGF1R) stimulate Wnt‐4 expression and β‐catenin activation in growth plate chondrocytes. The positive effects of IGF‐1/IGF1R on chondrocyte proliferation and terminal differentiation are partially inhibited by the Wnt antagonists sFRP3 and Dkk1. T₃ activates IGF‐1/IGF1R signaling and IGF‐1‐dependent PI3K/Akt/GSK‐3β signaling in growth plate chondrocytes undergoing proliferation and differentiation to prehypertrophy. T₃‐mediated Wnt‐4 expression, β‐catenin activation, cell proliferation, and terminal differentiation of growth plate chondrocytes are partially prevented by the IGF1R inhibitor picropodophyllin as well as by the PI3K/Akt signaling inhibitors LY294002 and Akti1/2. These data indicate that the interactions between thyroid hormone and β‐catenin signaling in regulating growth plate chondrocyte proliferation and terminal differentiation are modulated by IGF‐1/IGF1R signaling through both the Wnt and PI3K/Akt signaling pathways. While chondrocyte proliferation may be triggered by the IGF‐1/IGF1R‐mediated PI3K/Akt/GSK3β pathway, cell hypertrophy is likely due to activation of Wnt/β‐catenin signaling, which is at least in part initiated by IGF‐1 signaling or the IGF‐1‐activated PI3K/Akt signaling pathway. © 2010 American Society for Bone and Mineral Research     

Thyroid Hormone in the Treatment of Post-transplant Acute Tubular Necrosis (ATN)

Delayed graft function (DGF) in cadaver kidney transplants is a common problem and is often due to acute tubular necrosis (ATN). DGF in transplants may have a deleterious effect on long-term graft survival. Since thyroid hormone has been shown to hasten recovery from ATN in experimental models, we designed a trial to determine if a defined course of triiodothyronine (T3) would improve the short- or long-term outcome of patients with DGF in cadaveric transplants. A prospective, randomized, placebo controlled, double blind trial of T3 was carried out in patients with DGF in cadaveric renal transplants. End-points were percentage requiring dialysis, percentage recovering function, time to recovery and length of hospital stay. Long-term outcomes were percentage grafts functioning at 1 year and mean serum creatinine at 1 year. Forty-four patients were randomized to receive either T3 or placebo. Three patients were dropped from each group when early biopsies disclosed that DGF was due to rejection. The groups were well matched by age, cold ischemia time of the graft, and percentage reactivity to a random panel of antigens. Baseline thyroid function studies, including T3, reverse T3 (rT3), and thyroid stimulating hormone (TSH) levels, were similar between the two groups and typical of 'euthyroid-sick syndrome'. T3 had no effect on percentage requiring dialysis, time to recovery, percentage recovering function, or length of stay. At 1 year follow-up, graft function was similar in both groups and significantly lower than that seen in patients with good initial function. Thyroid hormone, given early in the course of DGF in cadaver kidney recipients, had no effect on the course of DGF. Long-term graft function is impaired in patients who experience post-transplant DGF compared to those who have good initial function.     

Activation of Wnt Planar cell polarity (PCP) signaling promotes growth plate column formation in vitro

Disrupting the Wnt Planar Cell Polarity (PCP) signaling pathway in vivo results in loss of columnar growth plate architecture, but it is unknown whether activation of this pathway in vitro is sufficient to promote column formation. We hypothesized that activation of the Wnt PCP pathway in growth plate chondrocyte cell pellets would promote columnar organization in these cells that are normally oriented randomly in culture. Rat growth plate chondrocytes were transfected with plasmids encoding the Fzd7 cell‐surface Wnt receptor, a Fzd7 deletion mutant lacking the Wnt‐binding domain, or Wnt receptor‐associated proteins Ror2 or Vangl2, and then cultured as three‐dimensional cell pellets in the presence of recombinant Wnt5a or Wnt5b for 21 days. Cellular morphology was evaluated using histomorphometric measurements. Activation of Wnt PCP signaling components promoted the initiation of columnar morphogenesis in the chondrocyte pellet culture model, as measured by histomorphometric analysis of the column index (ANOVA p = 0.01). Activation of noncanonical Wnt signaling through overexpression of both the cell‐surface Wnt receptor Fzd7 and receptor‐associated protein Ror2 with addition of recombinant Wnt5a promotes the initiation of columnar architecture of growth plate chondrocytes in vitro, representing an important step toward growth plate regeneration. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1906–1914, 2012     

胰岛素样生长因子和胰岛素样生长因子结合蛋白在软骨细胞生长和发育过程中的调控作用研究进展

胰岛素样生长因子（Insulin-like growth factors,IGFs）,是机体生理情况下调控生长和发育的重要生长因子。IGFs在长骨发育,即软骨内化骨的调控中发挥重要作用。IGF-I参与调节了间充质干细胞的成软骨过程并进而参与了软骨组织稳态的保持。胰岛素样生长因子结合蛋白（Insulin-like growth factor-binding proteins,IGFBPs）代表了一个能与IGF-I和IGF-2相结合的进化上保守的蛋白质家族,包括6个独立的家族成员IGFBP1、2、3、4、5、6,它们具有调控和储存转运IGFs的作用以及独立于IGFs的作用,在机体生长和发育中发挥重要的功能,在软骨组织的发育中同样扮演着重要的角色。本文综述了IGFs和IGFBPs对软骨细胞生长和发育的生理调节功能,从而为IGFs在软骨组织工程领域的应用提供参考。     

生长激素联合阿司匹林在体外受精-胚胎移植周期中的应用

目的:探讨生长激素(GH)联合阿司匹林在体外受精-胚胎移植(IVF-ET)中,对卵巢低反应患者促排卵周期的作用。方法:先前IVF促排卵周期中卵巢低反应患者40例,随机均分为联合用药组和对照组,在对照组应用促性腺激素释放激素激动剂(GnRH-a)和促性腺激素(Gn)促排卵的基础上,联合用药组还加用GH和小剂量的阿司匹林。比较两种不同治疗方法的作用效果。结果:联合用药组与对照组比较可明显提高卵泡数(P(0.01)、获卵数(P(0.01)、MⅡ期卵母细胞数(P(0.01)、受精率(P(0.05);而优质胚胎数没有明显差异(P(0.05),妊娠率提高不明显(P(0.05)。结论:IVF-ET的促排卵周期中,联合应用GH和阿司匹林可提高卵巢低反应患者的卵泡数、获卵数及受精率。     

Interactions of Prolactin and Growth Hormone (GH) in the Regulation of Mammary Gland Function and Epithelial Cell Survival

The relative importance of GH³ and prolactin in mammary gland function varies between species with prolactin playing a major role in rodents and GH taking lead role in ruminants. In rodents, however, GH appears to play a vital role in maintaining a high-fat/low volume milk in the absence of prolactin and a similar finding has been demonstrated in goats where prolactin deficiency causes a more modest (15%) decrease in milk yield. Surprisingly GH-deficiency in goats induced no further decline in milk yield whereas exogenous GH or prolactin both stimulated milk output considerably. Although direct effects of prolactin on mammary epithelial cells are well-documented effects of GH are believed to be mediated indirectly via IGF-1 production from the liver. We have been unable to confirm this hypothesis in rats and believe this to be because it is too simplistic. By considering prolactin and GH to be survival factors for the mammary gland we now propose a mechanism by which they interact through the IGF system. Involution of the mammary gland involves apoptosis and, in rats, it is induced by prolactin-deficiency or milk accumulation. Coincidentally with this process mammary epithelial cells synthesize and secrete an IGF binding protein, IGFBP-5. We hypothesize that GH stimulates IGF-1 production, possibly from the mammary parenchyma. IGF-1 then acts as a survival factor for the mammary gland. Prolactin plays an essential role since it suppresses the secretion of IGFBP-5 which would otherwise inhibit IGF-1 action and lead to the induction of cell death.     

A potential autocrine pathway for growth hormone releasing hormone (GHRH) and its receptor in human prostate cancer cell lines

BACKGROUND: Recent studies have shown that GHRH antagonists inhibit prostate tumour growth and IGF-II production both in vivo and in vitro. The mechanism underlying these observations is unknown, but may involve an interaction with a prostatic GHRH receptor (GHRH-R), raising the possibility of an autocrine pathway for the GHRH axis in the prostate. METHODS: GHRH and GHRH-R mRNA expression was examined by RT-PCR in human prostate cancer cell lines, and the authenticity of PCR products was confirmed by Southern analysis and cDNA sequencing. Immunohistochemical techniques were used to examine the expression of GHRH protein in prostate cancer cell lines. RESULTS: GHRH-R (mRNA) and GHRH (mRNA and protein) are co-expressed in the ALVA-41, DU145, LNCaP and PC3 human prostate cancer cell lines. CONCLUSIONS: These observations suggest the presence of an intact prostatic GHRH autocrine pathway which may stimulate prostate cell proliferation. This pathway may be disrupted by GHRH antagonists.     

Localization of Matrix Metalloproteinases, (MMPs) Their Tissue Inhibitors, and Vascular Endothelial Growth Factor (VEGF) in Growth Plates of Children and Adolescents Indicates a Role for MMPs in Human Postnatal Growth and Skeletal Maturation

Numerous studies have focused on the expression, regulation, and biological significance of matrix metalloproteinases (MMPs) in the growth plate. Findings in mouse knockout models and in vitro data from various species indicate that MMPs not only degrade extracellular matrix components but may regulate the activity of local growth factors. In this study we investigated the presence, distribution, and activity of various MMPs and inhibitors, tissue transglutaminase (tTG or TG2) and vascular endothelial growth factor (VEGF) in the human child and adolescent growth plates by means of immunohistochemistry and gelatin zymography. Tissue was derived during orthopedic surgery (epiphysiodesis) in two prepubertal and four pubertal patients.MMP-2 and MMP-14 were present in reserve cell chondrocytes. MMP-14 was the most prominent MMP within all zones of the growth plate including proliferating chondrocytes. MMP-1 and MMP-13 (collagenases 1 and 3), MMP-9 (gelatinases B), MMP-10, and MMP-11 (stromelysins) and VEGF were positive in hypertrophic chondrocytes and osteoblasts. MMP-2 showed the same expression pattern but was negative in osteoblasts. Osteoclasts stained positive for MMP-9, MMP-2, and TG2. Tissue inhibitor of MMP (TIMP)-1 was present in all zones of the growth plate, osteoblasts, and osteoclasts; TIMP-2 was found in hypertrophic chondrocytes and osteoblasts. In summary, the presence of MMPs, TIMPs, TG2, and VEGF in our study indicated that the MMPs are relevant in growth plate physiology during the postnatal period in humans. The specific location of MMP expression within the growth plate may be the basis for further studies on the role of MMPs in the local regulation of chondrocyte differentiation, proliferation, and ossification at the chondroosseus junction.     

Effects of 24 Months of Growth Hormone (GH) Treatment on Serum Carboxylated and Undercarboxylated Osteocalcin Levels in GH-Deficient Adults

We studied the effect of growth hormone (GH) replacement on bone mineral density (BMD) and some parameters of bone metabolism, including undercarboxylated osteocalcin (ucOC), an independent predictive marker of fracture risk, which has not been previously determined or compared during GH treatment. Measurements were performed at baseline and after 6, 12, 18 and 24 months of the initiation of the GH therapy in 21 adult patients with GH deficiency. Significant increases were observed in BMD after 1 year at the lumbar spine and after 1.5 years at the femoral neck. Serum total OC and carboxylated (c) OC increased and reached the maximum at 6 months, but the values remained over the baseline at both 12 and 18 months. The ucOC:total OC ratio changed contrarily: it decreased at 6 months, then increased again and reached the baseline level during the next 18 months. Serum calcium (Ca), phosphate (P) and total alkaline phosphatase (ALP) levels increased after 6 months, thereafter the Ca and P values decreased, while the total ALP remained elevated until 12 months. Serum parathormone decreased at 12 months and increased again thereafter. GH replacement therapy is associated with improvement of ucOC, a marker of fracture risk, which in addition to the increase of BMD, might contribute to the beneficial effect of GH replacement therapy on bone metabolism.     

Subcellular distribution of the insulin‐like growth factor (IGF) binding proteins (IGFBPs) 2 and 3 in articular chondrocytes

The insulin‐like growth factor (IGF) is a major anabolic regulator in articular cartilage. The IGF‐binding proteins (IGFBPs) are increased during osteoarthritis (OA), but the function of the later proteins remains unknown. In general, the IGFBPs are pluripotential effectors capable of IGF regulation and of acting on their own to control key cell functions, including survival and proliferation. The independent functions are often associated with their cell location, and therefore this study explores the distribution of IGFBP‐2 and IGFBP‐3 in articular chondrocytes. Immunohistochemistry was used to localize IGFBP‐2 in normal human articular cartilage. Bovine chondrocytes were used for subcellular fractionation (hypotonic cell lysis) under nonreducing conditions and nuclear purification (centrifugation on sucrose cushions). Cell fraction markers and IGFBPs were assayed in the subcellular fractions by Western immunoblot. The IHC results showed association of IGFBP‐2 with chondrocytes, but not with the nuclei. Subcellular fractionation of isolated chondrocytes yielded intact nuclei as assessed at the light microscopic level; the nuclear marker histone H1 was exclusively associated with this fraction. More than 90% of the cytoplasmic marker GAPDH and all the detectable IGFBP‐2 were in the cytoplasmic fraction. Immunoreactive IGFBP‐3 was found in the cytoplasmic and peri‐nuclear/nuclear fractions. Chondrocytes contain intracellular IGFBP‐2 and IGFBP‐3 but only IGFBP‐3 is associated with nuclei. This suggests the hypothesis that the actions of these IGFBPs in articular cartilage extend beyond the classic modulation of IGF receptor action. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1421–1427, 2008     

Effects of Growth Hormone Administration on Bone Mineral Metabolism,PTH Sensitivity and PTH Secretory Rhythm in Postmenopausal Women With Established Osteoporosis

Introduction : Growth hormone (GH) replacement improves target organ sensitivity to PTH, PTH circadian rhythm, calcium and phosphate metabolism, bone turnover, and BMD in adult GH‐deficient (AGHD) patients. In postmenopausal women with established osteoporosis, GH and insulin like growth factor‐1 (IGF‐1) concentrations are low, and administration of GH has been shown to increase bone turnover and BMD, but the mechanisms remain unclear. We studied the effects of GH administration on PTH sensitivity, PTH circadian rhythm, and bone mineral metabolism in postmenopausal women with established osteoporosis. Materials and Methods : Fourteen postmenopausal women with osteoporosis were compared with 14 healthy premenopausal controls at baseline that then received GH for a period of 12 mo. Patients were hospitalized for 24 h before and 1, 3, 6, and 12 mo after GH administration and half‐hourly blood and 3‐h urine samples were collected. PTH, calcium (Ca), phosphate (PO₄), nephrogenous cyclic AMP (NcAMP), β C‐telopeptide of type 1 collagen (βCTX), procollagen type I amino‐terminal propeptide (PINP), and 1,25‐dihydroxyvitamin D [1,25(OH)₂D] were measured. Circadian rhythm analysis was performed using Chronolab 3.0 and Student's t‐test and general linear model ANOVAs for repeated measures were used where appropriate. Results : IGF‐1 concentration was significantly lower in the women with established osteoporosis compared with controls (101.5 ± 8.9 versus 140.9 ± 10.8 μg/liter; p < 0.05) and increased significantly after 1, 3, 6, and 12 mo of GH administration (p < 0.001). Twenty‐four‐hour mean PTH concentration was higher in the osteoporotic women (5.4 ± 0.1 pM) than in healthy controls (4.4 ± 0.1 pM, p < 0.001) and decreased after 1 (5.2 ± 0.1 pM, p < 0.001), 3 (5.0 ± 0.1 pM, p < 0.001), 6 (4.7 ± 0.1 pM, p < 0.001), and 12 mo (4.9 ± 0.1 pM, p < 0.05) of GH administration compared with baseline. NcAMP was significantly lower in osteoporotic women (17.2 ± 1.2 nM glomerular filtration rate [GFR]) compared with controls (21.4 ± 1.4 nM GFR, p < 0.05) and increased after 1 (24.2 ± 2.5 nM GFR, p < 0.05), 3 (27.3 ± 1.5 nM GFR, p < 0.001), and 6 mo (32.4 ± 2.5 nM GFR, p < 0.001) compared with baseline. PTH secretion was characterized by two peaks in premenopausal women and was altered in postmenopausal women with a sustained increase in PTH concentration. GH administration also restored a normal PTH secretory pattern in the osteoporotic women. The 24‐h mean adjusted serum calcium (ACa) concentration increased at 1 and 3 mo (p < 0.001) and PO₄ at 1, 3, 6, and 12 mo (p < 0.001). 1,25(OH)₂D concentration increased after 3, 6, and 12 mo of GH (p < 0.05). An increase in urine Ca excretion was observed at 3 and 6 mo (p < 0.05), and the renal threshold for maximum tubular phosphate reabsorption rate (TmPO4/GFR) increased after 1, 3, 6, and 12 mo (p < 0.05). βCTX concentration increased progressively from 0.74 ± 0.07 μg/liter at baseline to 0.83 ± 0.07 μg/liter (p < 0.05) at 1 mo and 1.07 ± 0.09 μg/liter (p < 0.01) at 3 mo, with no further increase at 6 or 12 mo. PINP concentration increased progressively from baseline (60 ± 5 μg/liter) to 6 mo (126 ± 11 μg/liter, p < 0.001), with no further increase at 12 mo. The percentage increase in PINP concentration was significantly higher than βCTX (p < 0.05). Conclusions : Our study shows that GH has a regulatory role in bone mineral metabolism. GH administration to postmenopausal osteoporotic women improves target organ sensitivity to PTH and bone mineral metabolism and alters PTH secretory pattern with greater increases in bone formation than resorption. These changes, resulting in a net positive bone balance, may partly explain the mechanism causing the increase in BMD after long‐term administration of GH in postmenopausal women with osteoporosis shown in previous studies and proposes a further component in the development of age‐related postmenopausal osteoporosis.     

Regulation of prostate-specific antigen (PSA) gene expression and release in LNCaP prostate cancer by antagonists of growth hormone-releasing hormone and vasoactive intestinal peptide

BACKGROUND: Prostate-specific antigen (PSA) is the best tumor marker for diagnosis and prognosis of prostatic carcinoma. The secretion of PSA from LNCaP human prostate cancer cells is influenced by acute stimuli such as vasoactive intestinal peptide (VIP), growth hormone-releasing hormone (GHRH), and chronic stimuli like androgens. METHODS: To study the regulation of basal and VIP/GHRH or androgen-stimulated secretion from LNCaP cells, we used a superfusion system, which allowed us to simultaneously measure PSA gene expression, PSA secretion, and cAMP release from the same cancer cells. LNCaP cancer cells were also implanted orthotopically into nude mice. RESULTS: VIP (30 pM-3 nM), GHRH (3 nM-300 nM), and dihydrotestosterone (100 nM) induced a significant increase in PSA gene expression, PSA secretion, and cAMP release. The dose and time-dependent effects of peptides were manifested only in the presence of androgens. At the end of continuous stimulation of cells with 1 nM VIP for 2 hr, large amounts of stored immunoreactive PSA still remained in the cells. Adenylate cyclase activator, forskolin (FSK), significantly increased PSA secretion and gene expression, and potassium, which causes nonspecific depolarization of membranes, augmented gene expression, and secretion of PSA, but did not influence cAMP release. This suggests that PSA secretion is regulated by cAMP-dependent as well as cAMP-independent pathways. In superfusion system, stimulatory effects of VIP and GHRH on PSA secretion were inhibited by VIP antagonist JV-1-53, and less by GHRH antagonist JV-1-38. In cell cultures, JV-1-38 had a stronger inhibitory effect on proliferation, indicating an involvement of the recently discovered tumoral GHRH receptors in this process. In nude mice, with orthotopically implanted LNCaP cancer cells, GHRH antagonist JV-1-38 alone or androgen ablation by castration had no effect on tumor growth and PSA levels. However, castration combined with treatment with GHRH antagonist, significantly decreased tumor growth and PSA secretion. CONCLUSIONS: Our findings suggest that the secretion of PSA is regulated rather than constitutive, contrary to previous reports. In addition, the effect of GHRH and VIP antagonists on PSA secretion from prostate cancer cells is not correlated with their antiproliferative action.     

Comparison of the Abilities of Human Parathyroid Hormone (hPTH)-(1-34) and [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 to Stimulate Femoral Trabecular Bone Growth in Ovariectomized Rats

hPTH-(1-31)NH₂, so far the smallest of the potently anabolic N-terminal fragments of the human parathyroid hormone, stimulates trabecular growth in the distal femurs of ovariectomized (OVX) rats as strongly as hPTH-(1-34) when injected at a high daily dose such as 1 nmol/100 g of body weight, but it is only about 70% as effective as hPTH-(1-34) when injected at the suboptimal 0.6 nmol/100 g of body weight. A lactam derivative of hPTH-(1-31)-NH₂, [Leu²⁷]-cyclo(Glu²²-Lys²⁶)-hPTH-(1-31)NH₂, is a much more effective stimulator of adenylyl cyclase in ROS 17/2 rat osteoblast-like cells and a significantly more effective stimulator of femoral trabecular growth in OVX rats than hPTH-(1-31)NH₂. We have now shown that [Leu²⁷]-cyclo(Glu²²-Lys²⁶)-hPTH-(1-31)NH₂ prevents the OVX-induced loss of femoral trabeculae significantly more effectively than hPTH-(1-34) and stimulates the thickening of the trabeculae remaining in severely depleted femoral trabecular bone of OVX rats as effectively as hPTH-(1-34) when injected at 0.6 nmol/100 g of body weight. Received: 26 December 1997 / Accepted: 23 March 1998