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Insulin-like growth factor-1 (IGF-1) is a critical modulator of cell growth and survival, making it a central part of maintaining essentially every biological system in the body. Knowledge of the intricate mechanisms involved in activating IGF-1 signaling is not only key to understanding basic processes of growth and development, but also for addressing diseases, such as cancer and diabetes. This brief review explores how dysregulation of normal IGF-1 signaling can impact growth by examining its role in postnatal bone elongation. IGF-1 actions are dysregulated in autoimmune diseases, such as juvenile idiopathic arthritis and chronic kidney disease, which results in growth stunting. Conversely, childhood obesity results in growth acceleration, premature growth cessation, and ultimately, diminished bone quality, while systemic IGF-1 levels remain normal. Understanding the role of IGF-1 signaling in normal and dysregulated growth can add to other studies that address how this system regulates chronic diseases. [ABSTRACT FROM AUTHOR]

Dexamethasone (Dex) is a type of glucocorticoid drug. Long term use can induce growth plate chondrocytes (GPCs) apoptosis, impair differentiation, and inhibit cell proliferation and bone growth. It has been reported that Krüppel-like factor 2 (KLF2) inhibits osteoblast damage induced by Dex, but the role in Dex-induced GPCs remains unclear. Dex was used to construct a model of growth plate injury in vitro. CCK-8 and TUNEL kits were used to determine cell viability and apoptosis. A model of growth plate injury was established by intraperitoneal injection of Dex. Immunohistochemistry was used to investigate the expression of KLF2 in rats. The results showed that KLF2 expression of rat tibial GPCs was down-regulated after Dex stimulation. Overexpression of KLF2 promoted cell viability and cell cycle, while inhibited apoptosis of growth plate Dex-induced chondrocytes. Moreover, KLF2 inhibited Runx2-mediated PI3K/AKT and ERK signalling pathways. And PI3K/AKT and ERK signalling pathways, which were involved in the regulation of KLF2 on GPCs. Further studies showed that KLF2 alleviated growth plate injury in vivo. In conclusion, our study found that KLF2 promoted proliferation and inhibited apoptosis of Dex-induced GPCs by targeting the Runx2-mediated PI3K/AKT and ERK signalling pathways.

Dexamethasone (Dex) is a type of glucocorticoid drug. Long term use can induce growth plate chondrocytes (GPCs) apoptosis, impair differentiation, and inhibit cell proliferation and bone growth. It has been reported that Krüppel-like factor 2 (KLF2) inhibits osteoblast damage induced by Dex, but the role in Dex-induced GPCs remains unclear. Dex was used to construct a model of growth plate injury in vitro. CCK-8 and TUNEL kits were used to determine cell viability and apoptosis. A model of growth plate injury was established by intraperitoneal injection of Dex. Immunohistochemistry was used to investigate the expression of KLF2 in rats. The results showed that KLF2 expression of rat tibial GPCs was down-regulated after Dex stimulation. Overexpression of KLF2 promoted cell viability and cell cycle, while inhibited apoptosis of growth plate Dex-induced chondrocytes. Moreover, KLF2 inhibited Runx2-mediated PI3K/AKT and ERK signalling pathways. And PI3K/AKT and ERK signalling pathways, which were involved in the regulation of KLF2 on GPCs. Further studies showed that KLF2 alleviated growth plate injury in vivo. In conclusion, our study found that KLF2 promoted proliferation and inhibited apoptosis of Dex-induced GPCs by targeting the Runx2-mediated PI3K/AKT and ERK signalling pathways. [ABSTRACT FROM AUTHOR]

Dexamethasone (Dex) is reported to cause bone growth retardation in children, which is associated with the increased apoptosis and decreased proliferation of growth plate chondrocytes. Sirtuin 1 (SIRT1) plays an important role in chondrocyte function and homeostasis. Thus, we further explored the regulatory mechanism of SIRT1 in Dex‐induced growth plate chondrocyte dysfunction. SIRT1 expression was detected in Dex‐treated growth plate chondrocytes using RT‐qPCR and western blot assay. The modulation of SIRT1 on SOX2 expression was evaluated. Besides, we identified that SIRT1 was targeted by miR‐211‐5p using TargetScan and RNA pull‐down assay. A loss‐of‐function assay was performed to evaluate the effects of miR‐211‐5p on Dex‐induced growth plate chondrocyte dysfunction in vitro and in vivo. We found that SIRT1 was downregulated in Dex‐treated growth plate chondrocytes. The expression of SOX2 was upregulated by overexpression SIRT1. Meanwhile, downregulation of SOX2 weakened the positive function of SIRT1 overexpression on Dex‐induced growth plate chondrocytes dysfunction. Subsequently, we confirmed that SIRT1 was targeted by miR‐211‐5p. MiR‐211‐5p inhibitor increased the expression levels of SIRT1 and SOX2, and restored the Dex‐treated growth plate chondrocyte function. Animal assays further demonstrated that the effects of miR‐211‐5p on the growth plate chondrogenesis. In conclusion, our data suggest that SIRT1 exerts a protective effect on growth plate chondrocyte under Dex stimulation. MiR‐211‐5p/SIRT1/SOX2 axis regulates the process of Dex‐inhibited growth plate chondrogenesis. [ABSTRACT FROM AUTHOR]

BACKGROUND: Epiphyseal closure indicates the long bones stop growing, which is caused by the apoptosis of growth plate chondrocytes. It has been reported that aromatase inhibitors can inhibit the production of estrogen, and the cessation of bone growth is caused by epiphyseal closure. Therefore, it has been inferred that aromatase inhibitors can inhibit growth plate chondrocyte apoptosis and delay epiphyseal closure. OBJECTIVE: To compare the effects of several aromatase inhibitors (resveratrol, biochanin A, and flax lignans) on inhibiting the apoptosis of rat growth plate chondrocyte and to verify the effect of resveratrol on delaying epiphyseal closure and promoting the growth of long bones in rats. METHODS: Rat tibial and femoral growth plate cartilage samples were dissected, and growth plate chondrocytes were isolated and cultured in vitro by two-step enzyme digestion. Chondrocytes were morphologically observed and identified by inverted phase contrast microscope and type II collagen immunofluorescence experiments. Then interleukin 1β was used to induce apoptosis of growth plate chondrocytes. Cells were cultured with culture medium containing 10, 20, 40, and 60 μmol/L resveratrol, with culture medium containing 5, 10, 20, 30 μmol/L biochanin, or with culture medium containing 1, 10, 20, 40 μmol/L flax lignans to compare the effects of different concentrations of drugs on the apoptosis of growth plate chondrocytes. Resveratrol which had the best effect was administered to rats by gavage to investigate its effect on the epiphyseal closure of rat tibial plateau and the growth of long bones. RESULTS AND CONCLUSION: Appropriate concentrations of resveratrol, biochanin A, and flax lignans could inhibit the apoptosis of growth plate chondrocytes to varying degrees. The best drug concentrations for inhibiting cell apoptosis were 40, 10, and 20 μmol/L in sequence, and 40 μmol/L resveratrol had the best effect on inhibiting the apoptosis of growth plate chondrocytes. Administration of 40 mg/kg resveratrol by gavage could delay epiphyseal closure of tibial plateau and promote the growth of long bones in rats. Therefore, resveratrol can promote bone growth during developmental period by prolonging the time of bone growth. [ABSTRACT FROM AUTHOR]

Short stature is the second conspicuous characteristic of osteogenesis imperfecta (OI), but the etiological mechanism is unclear. The proliferation of growth plate chondrocytes (GPCs) plays an essential role in longitudinal bone growth, and chondrocyte division deficiency can cause shortened limbs. However, few studies have reported the abnormal changes of growth plate and GPCs in OI. In this study, the cell proliferative performance of GPCs in heterozygous Col1a2 oim/+ mice were studied and the underlying mechanism was explored by RNA-Sequencing. The results indicated that chondrocytes of Col1a2 oim/+ background displayed impaired cell division when compared with cells of wild-type littermates. A group of differentially expressed genes involving chondrocyte proliferation related pathways including cell cycle, TGF-β signaling pathway and Hedgehog signaling pathway were identified. These dysregulated genes and pathways in GPCs of Col1a2 oim/+ mice are likely to play an important role in their shortened long bones. Further investigations to reveal the effect of these genes on bone elongation not only facilitate the understanding of OI short stature, but also contribute to developing new treatments. • The proliferation of growth plate chondrocytes was significantly impaired in a mouse model of OI. • The DEGs were involved in cell cycle, TGF-β signaling pathway and Hedgehog signaling pathway in OI growth plate chondrocytes. • These dysregulated genes and pathways in chondrocytes might be associated with the their bone growth deficiency of OI mice. [ABSTRACT FROM AUTHOR]

Menisci are wedge‐shaped cartilage discs that are divided into two parts: the avascular and vascular regions. They are formed by fibrocartilage tissue, which contains round cartilage‐like cells and extracellular matrix. Meniscus injury in animals is a common orthopedic problem, but data on the natural healing process mainly deals with the vascular zone. The healing processes in the avascular zone of the meniscus are significantly limited. Thus, this study aimed to evaluate autologous growth plate chondrocytes' impact on the healing process of a damaged meniscus in the avascular zone based on a growing animal model. The study group consisted of 10 pigs at about three months of age. From each animal, chondrocytes from the iliac growth plate and from concentrated bone marrow were taken. Knee joints were divided into right (R) and left (L). The medial meniscus of the R knee joint was treated with a hyaluronic acid based scaffold incubated with bone marrow cells from marrow aspirates (nCHON). The medial meniscus of the L knee joint was treated with a hyaluronic acid based scaffold incubated with bone marrow cells from marrow aspirates supplemented with immature chondrocytes isolated from growth plates (wCHON). The meniscus was damaged in the avascular zone in both knee joints. Followingly, the damaged part of the meniscus was filled with a scaffold with cells from the concentrated bone marrow and from growth plate chondrocytes. In the control group, a scaffold with concentrated bone marrow cells was used. After three months the animals were euthanized and preparations (microscopic slides) were made from the meniscus' damaged part. A qualitative and quantitative analysis have been prepared. The wCHON group in comparison with the nCHON group showed a statistically significantly higher number of fusiform cells on the surface of the graft as well as better healing of the graft. In addition, the degree of vascularization was higher in specimens from the wCHON group than in the nCHON group. The results of our research on immature pig knees revealed that mesenchymal stem cell and growth plate chondrocytes could be treated as the cell source for meniscus reconstruction, and growth plate chondrocytes enhance healing processes in the avascular zone of the injured meniscus. [ABSTRACT FROM AUTHOR]

Several studies have demonstrated the core circadian rhythm gene Bmal1 could regulate the clock control genes (CCGs) expression and maintain the integrity in cartilage tissue. In addition, its abnormal expression is connected with the occurrence and development of several diseases including osteoarthritis (OA). However, the relationship between Bmal1 and cartilage development still needs to be fully elucidated. Here, we bred tamoxifen-induced cartilage-specific knockout mice to learn the effects of Bmal1 on the cartilage development and its underlying mechanisms at specific time points. We observed that Bmal1 ablated mice showed growth retardation during puberty, and the length of whole growth plate and the proliferation zone were both shorter than those in the control group. Deletion of Bmal1 significantly inhibited the chondrocytes proliferation and activated cells apoptosis in the growth plate. Meanwhile, knockout of Bmal1 attenuated the expression of VEGF and HIF1α and enhanced the level of MMP13 and Runx2 in the growth plate chondrocytes. Consistent with these findings in vivo, ablation of Bmal1 could also lead to decrease chondrocytes proliferation, the expression of HIF1α and VEGF and elevate apoptosis in cultured chondrocytes. These findings suggest that Bmal1 plays a pivotal role in cartilage development by regulating the HIF1α-VEGF signaling pathway. [ABSTRACT FROM AUTHOR]

ABSTRACT Autophagy is activated during nutritionally depleted or hypoxic conditions to facilitate cell survival. Because growth plate is an avascular and hypoxic tissue, autophagy may have a crucial role during chondrogenesis; however, the functional role and underlying mechanism of autophagy in regulation of growth plate remains elusive. In this study, we generated TamCartAtg7-/- (Atg7cKO) mice to explore the role of autophagy during endochondral ossification. Atg7cKO mice exhibited growth retardation associated with reduced chondrocyte proliferation and differentiation, and increased chondrocyte apoptosis. Meanwhile, we observed that Atg7 ablation mainly induced the PERK-ATF4-CHOP axis of the endoplasmic reticulum (ER) stress response in growth plate chondrocytes. Although Atg7 ablation induced ER stress in growth plate chondrocytes, the addition of phenylbutyric acid (PBA), a chemical chaperone known to attenuate ER stress, partly neutralized such effects of Atg7 ablation on longitudinal bone growth, indicating the causative interaction between autophagy and ER stress in growth plate. Consistent with these findings in vivo, we also observed that Atg7 ablation in cultured chondrocytes resulted in defective autophagy, elevated ER stress, decreased chondrocytes proliferation, impaired expression of col10a1, MMP­13, and VEGFA for chondrocyte differentiation, and increased chondrocyte apoptosis, while such effects were partly nullified by reduction of ER stress with PBA. In addition, Atg7 ablation-mediated impaired chondrocyte function (chondrocyte proliferation, differentiation, and apoptosis) was partly reversed in CHOP-/- cells, indicating the causative role of the PERK-ATF4-CHOP axis of the ER stress response in the action of autophagy deficiency in chondrocytes. In conclusion, our findings indicate that autophagy deficiency may trigger ER stress in growth plate chondrocytes and contribute to growth retardation, thus implicating autophagy as an important regulator during chondrogenesis and providing new insights into the clinical potential of autophagy in cartilage homeostasis. © 2017 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

ABSTRACT Yes-associated protein (YAP) and extracellular signal-regulated kinase (ERK) have been considered as key regulators in tissue homeostasis, organ development, and tumor formation. However, the roles of YAP and ERK in the mediating strain mechanosensing in the growth plate cartilage have not been determined. In this study, chondrocytes obtained from the growth plate cartilage of 2-week-old Sprague-Dawley rats were subjected to the mechanical strain with different magnitudes and durations at a frequency of 0.5 Hz. We found that YAP and ERK activation in response to mechanical strain was time and magnitude dependent. Pretreatment with a RhoA inhibitor (C3 toxin) or a microfilament cytoskeleton disrupting reagent (cytochalasin D) could suppress their activation. In addition, activated YAP and ERK were able to induce cell cycle progression by up-regulating the expression of cell cycle-related genes. These results shed new light on the function of YAP and ERK in mechanical strain-promoted growth plate development. Our results also provided evidence that RhoA and cytoskeletal dynamics are required for this mechanotransduction. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1121-1129, 2016. [ABSTRACT FROM AUTHOR]

Abstract: Sex steroids are important regulators for longitudinal growth, bone mass accrual, and sexual dimorphism of the skeleton. 17β-Estradiol regulates proliferation and differentiation of female chondrocytes via a membrane-associated signaling pathway in addition to its estrogen receptor (ER) mediated effects. In contrast, testosterone does not elicit a similar membrane response, either in male or female cells. Whereas female rat growth plate chondrocytes convert testosterone to 17β-estradiol, male chondrocytes produce 5α-dihydrotestosterone (DHT). Previously DHT was found to mediate sex-specific effects of testosterone in male cells, but it is not known if a membrane-signaling pathway is involved. In this study, we hypothesized that DHT can induce sex-specific rapid membrane effects similar to other steroid hormones. Confluent cultures of chondrocytes isolated from resting zones of growth plates of both male and female rats were treated with 10−10–10−7 M testosterone or DHT for 3, 9, 90 and 270min and protein kinase C (PKC) and phospholipase A2 (PLA2) activities were measured. To examine potential signaling pathways involved in PKC activation, male chondrocytes were treated with 10−7 M DHT for 9min in the presence or absence of the phospholipase C (PLC) inhibitor U73122, the secretory PLA2 inhibitor quinacrine or the cytosolic PLA2 inhibitor AACOCF3; the Gαi inhibitor pertussis toxin (PTX) or Gαs activator cholera toxin (CTX), and the general G-protein inhibitor GDPβS; thapsigargin, an inhibitor of a Ca-ATPase pump in the endoplasmic reticulum; verapamil and nifedipine, inhibitors of specific L type Ca2+ channels on the cell membrane; and cyproterone acetate (CPA), which is an inhibitor of the classical androgen receptor (AR); as well as the transcription inhibitor actinomycin D, or the translation inhibitor cycloheximide. DHT induced a dose-dependent increase in PKC and PLA2 activity in male cells with the highest increase at 10−7 M DHT (p <0.05), whereas testosterone had no effect. PKC activity was augmented at 9 and 90min, and then decreased to baseline at 270min. Neither testosterone nor DHT affected PKC in female cells. U73122, quinacrine, and AACOCF3 inhibited DHT-induced activation of PKC. DHT treatment for 9min had no effect in [3H]-thymidine incorporation in quiescent confluent cultures but caused a dose dependent increase in alkaline phosphatase specific activity. Inhibition of PLC reduced the response of to DHT in a dose dependent manner, indicating that PLC is involved. In conclusion, our study indicates that DHT, but not testosterone, has sex-specific rapid membrane effects in male growth plate chondrocytes involving PLC and PLA2-mediated PKC signaling pathways. Together with previous observations showing that male cells convert testosterone to DHT, these results suggest that DHT might act in the membrane through an autocrine/paracrine mechanism. [Copyright &y& Elsevier]

Abstract: 1α,25-Dihydroxyvitamin D3 (1,25D3) regulates musculoskeletal cells via two different mechanisms: vitamin D receptor (VDR)-dependent gene transcription and rapid membrane-signaling via VDR as well as protein disulfide isomerase, family A, member 3 (Pdia3). In chondrocytes from the costochondral cartilage growth zone (GC), ligand binding to Pdia3 causes a rapid increase in phospholipase A2 (PLA2) activity leading to release of arachidonic acid and formation of lysophospholipid (LPL). LPL activates phospholipase C (PLC), and resulting inositol trisphosphate (IP3) and diacylglycerol contribute to PKCα activation and downstream activation of ERK1/2. PLA2 activating protein (PLAA) is increased in the growth zone of rat growth plates suggesting that it mediates the 1,25D3-dependent pathway. This study examined the role of PLAA in mediating 1,25D3-dependent PKC activation using GC cells and MC3T3-E1 wild-type and PLAA-silenced osteoblasts as models. PLAA, Pdia3, and caveolin-1 (Cav-1) were detected in plasma membranes and caveolae of GC and MC3T3-E1 cells. Pdia3-immunoprecipitated samples were positive for PLAA only after 1,25D3 treatment. Cav-1 was detected when immunoprecipitated with anti-Pdia3 and anti-PLAA in both vehicle and 1,25D3 treated cells. These observations were confirmed by immunohistochemistry. 1,25D3 failed to activate PLA2 and PKC or cause PGE2 release in PLAA-silenced cells. PLAA-antibody successfully blocked the PLAA protein and consequently suppressed PKC activity in GC and MC3T3-E1 cells. Crosslinking studies confirmed the localization of PLAA on the extracellular face on the plasma membrane in untreated MC3T3-E1 cells. Taken together, our results suggest that PLAA is an important mediator of 1α,25(OH)2D3 rapid membrane mediated signaling. 1α,25(OH)2D3 likely causes conformational changes bringing Pdia3 into proximity with PLAA, and aiding in transducing the signal from caveolae to the plasma membrane. [Copyright &y& Elsevier]

Abstract: Extracellular inorganic phosphate (Pi) concentrations are the highest in the growth plate just before the onset of mineralization. The study reported here demonstrates that Pi not only is required for hydroxyapatite mineral formation but also modulates terminal differentiation and apoptosis of growth plate chondrocytes. Extracellular Pi stimulated terminal differentiation marker gene expression, including the progressive ankylosis gene (ank), alkaline phosphatase (APase), matrix metalloproteinase-13 (MMP-13), osteocalcin, and runx2, mineralization, and apoptosis of growth plate chondrocytes. The stimulatory effect of extracellular Pi on terminal differentiation and apoptosis events of growth plate chondrocytes was dependent on the concentration, the expression levels of type III Na+/Pi cotransporters, and ultimately Pi uptake. A high extracellular Pi concentration was required for the stimulation of apoptosis, whereas lower Pi concentrations were required for the most effective stimulation of terminal differentiation events, including terminal differentiation marker gene expression and mineralization. Suppression of Pit-1 was sufficient to inhibit the stimulatory effects of extracellular Pi on terminal differentiation events. On the other hand, increasing the local extracellular Pi concentration by overexpressing ANK, a protein transporting intracellular PPi to the extracellular milieu where it is hydrolyzed to Pi in the presence of APase, resulted in marked increases of hypertrophic and early terminal differentiation marker mRNA levels, including APase, runx2 and type X collagen, and slight increase of MMP-13 mRNA levels, but decreased osteocalcin mRNA level, a late terminal differentiation markers. In the presence of levamisole, a specific APase inhibitor to prevent hydrolysis of extracellular PPi to Pi, ANK overexpression of growth plate chondrocytes resulted in decreased mRNA levels of hypertrophic and terminal differentiation markers but increased MMP-13 mRNA levels. In conclusion, with extracellular PPi inhibiting and extracellular Pi stimulating hypertrophic and terminal differentiation events, a precise regulation of PPi/Pi homeostasis is required for the spatial and temporal control of terminal differentiation events of growth plate chondrocytes. [Copyright &y& Elsevier]

The article discusses a study which examined the role of insulin-like growth factor 1 (IGF-1) signaling on the interaction between thyroid hormone and the Wnt/ß-catenin signaling pathways to regulate the proliferation and differentiation of growth plate chondrocytes. Results of the study showed that IGF-1 and the IGF-1 receptor stimulated the expression of Wnt-4 and the activation of ß-catenin in growth plate chondrocytes.

The article reports on thee results of research which was conducted in an effort to determine whether carboxypeptidase z modulates Wnt/Β-catenin signaling and terminal differentiation of growth plate cartilage. Researchers found that carboxypeptidase z links thyroid hormones and Wnt signaling pathways in growth plate chondrocytes and that thyroid hormones may regulate the differentiation of chondrocytes by regulating Wnt pathways.

Abstract: Growth plate chondrocytes produce proteoglycan-rich type II collagen extracellular matrix (ECM). During cell maturation and hypertrophy, ECM is reorganized via a process regulated by 1α,25(OH)2D3 and involving matrix metalloproteinases (MMPs), including MMP-3 and MMP-2. 1α,25(OH)2D3 regulates MMP incorporation into matrix vesicles (MVs), where they are stored until released. Like plasma membranes (PM), MVs contain the 1α,25(OH)2D3-binding protein ERp60, phospholipase A2 (PLA2), and caveolin-1, but appear to lack nuclear Vitamin D receptors (VDRs). Chondrocytes produce 1α,25(OH)2D3 (10−8 M), which binds ERp60, activating PLA2, and resulting lysophospholipids lead to MV membrane disorganization, releasing active MMPs. MV MMP-3 activates TGF-β1 stored in the ECM as large latent TGF-β1 complexes, consisting of latent TGF-β1 binding protein, latency associated peptide, and latent TGF-β1. Others have shown that MMP-2 specifically activates TGF-β2. TGF-β1 regulates 1α,25(OH)2D3-production, providing a mechanism for local control of growth factor activation. 1α,25(OH)2D3 activates PKCα in the PM via ERp60-signaling through PLA2, lysophospholipid production, and PLCβ. It also regulates distribution of phospholipids and PKC isoforms between MVs and PMs, enriching the MVs in PKCζ. Direct activation of MMP-3 in MVs requires ERp60. However, when MVs are treated with 1α,25(OH)2D3, PKCζ activity is decreased and PKCα is unaffected, suggesting a more complex feedback mechanism, potentially involving MV lipid signaling. [Copyright &y& Elsevier]

Thyroid hormone was first identified as a potent regulator of skeletal maturation at the growth plate more than forty years ago. Since that time, many in vitro and in vivo studies have confirmed that thyroid hormone regulates the critical transition between cell proliferation and terminal differentiation in the growth plate, specifically the maturation of growth plate chondrocytes into hypertrophic cells. However these studies have neither identified the molecular mechanisms involved in the regulation of skeletal maturation by thyroid hormone, nor demonstrated how the systemic actions of thyroid hormone interface with the local regulatory milieu of the growth plate. This article will review our current understanding of the role of thryoid hormone in regulating the process of endochondral ossification at the growth plate, as well as what is currently known about the molecular mechanisms involved in this regulation. [ABSTRACT FROM AUTHOR]

Abstract: Both male and female rat growth plate cartilage cells possess estrogen receptors (ERs), but 17β-estradiol (E2) activates protein kinase C (PKC) and PKC-dependent biological responses to E2 only in cells from female animals. PKC signaling can elicit genomic responses via mitogen activated protein kinase (MAPK) and E2 has been shown to activate ERK MAPK in many cells, suggesting that MAPK may play a role in growth plate chondrocytes as well. We tested if E2 increases MAPK activity and if so, whether the response is limited to female cells, if it is PKC-dependent, and if the mechanism involves traditional ER pathways. We also determined the contribution of MAPK to the biological response of growth plate chondrocytes and assessed the relative contributions of ERK, p38 and JNK MAPKs. Female rat costochondral cartilage cells were treated with E2 and MAPK-specific activity determined in cell layer lysates. The mechanism of MAPK activation was determined by treating the cells with E2 conjugated to bovine serum albumin (E2-BSA) to assess if membrane receptors were involved; stereospecificity was determined using 17α-estradiol; PKC and phospholipase C (PLC) dependence was determined using specific inhibitors; and the ER agonist diethylstilbestrol, the ER antagonist ICI 182780, and tamoxifen were used to assess the role of traditional ER pathways. E2 regulation of ERK1/2 MAPK was assessed and the relative roles of ERK1/2, p38 and JNK MAPKs determined using specific inhibitors. E2 caused a rapid dose-dependent activation of MAPK that was greatest in cells treated for 9 min with 10−9 M hormone; activity remained elevated for 3 h. E2''s effect on MAPK was stereospecific and comparable to that of E2-BSA. It was insensitive to DES and ICI 182780, dependent on PKC and PLC, blocked by tamoxifen and it did not require gene transcription or translation. E2 had no effect on ERK1 or ERK2 mRNA or protein but it caused a rapid phosphorylation of ERK1/2 at 9 min. Inhibition of ERK1/2 and p38 MAPK reduced the stimulatory effects of E2 on alkaline phosphatase activity and [35S]-sulfate incorporation. These results suggest that E2 regulates MAPK through a sex-specific membrane-mediated mechanism that does not involve cytosolic ERs in a traditional sense and that ERK1/2 and p38 mediate the downstream biological effects of the hormone. [Copyright &y& Elsevier]

Disturbances of the somatotropic hormone axis play an important pathogenic role in growth retardation and catabolism in children with chronic renal failure (CRF). The apparent discrepancy between normal or elevated growth hormone (GH) levels and diminished longitudinal growth in CRF has led to the concept of GH insensitivity, which is caused by multiple alterations in the distal components of the somatotropic hormone axis. Serum levels of IGF-I and IGF-II are normal in preterminal CRF, while in end-stage renal disease (ESRD) IGF-I levels are slightly decreased and IGF-II levels slightly increased. In view of the prevailing elevated GH levels in ESRD, these serum IGF-I levels appear inadequately low. Indeed, there is both clinical and experimental evidence for decreased hepatic production of IGF-I in CRF. This hepatic insensitivity to the action of GH may be partly the consequence of reduced GH receptor expression in liver tissue and partly a consequence of disturbed GH receptor signaling. The actions and metabolism of IGFs are modulated by specific high-affinity IGFBPs. CRF serum has an IGF-binding capacity that is increased by seven- to tenfold, leading to decreased IGF bioactivity of CRF serum despite normal total IGF levels. Serum levels of intact IGFBP-1, -2, -4, -6 and low molecular weight fragments of IGFBP-3 are elevated in CRF serum in relation to the degree of renal dysfunction, whereas serum levels of intact IGFBP-3 are normal. Levels of immunoreactive IGFBP-5 are not altered in CRF serum, but the majority of IGFBP-5 is fragmented. Decreased renal filtration and increased hepatic production of IGFBP-1 and -2 both contribute to high levels of serum IGFBP. Experimental and clinical evidence suggests that these excessive high-affinity IGFBPs in CRF serum inhibit IGF action in growth plate chondrocytes by competition with the type 1 IGF receptor for IGF binding. These data indicate that growth failure in CRF is mainly due to functional IGF deficiency. Combined therapy with rhGH and rhIGF-I is therefore a logical approach. [ABSTRACT FROM AUTHOR]

Chondrocytes in the growth plate undergo rapid proliferation during the process of enchondral ossification. The factors that regulate this proliferative activity have not been defined although several local autocrine or paracrine growth factors have recently been discovered in cartilage. Transforming growth factor-β (TGF-β) is an important regulator of cell metabolism and growth and is present in cartilage. The present study was designed to examine the influence of TGF-β on DNA synthesis in chick epiphyseal chondrocytes. Chondrocytes were plated in serum-free (BSA-supplemented) medium or medium containing 10% FBS, and after 24 hours in monolayer culture, were treated with TGF-β caused a biphasic dose-dependent increase in thymidine incorporation. The effect was greatly increased in serum-containing cultures where a maximal 20-fold increase in thymidine incorporation occurred compared with the 21/2-fold maximal increase obtained in serum-free cultures. The effect was present by 12 hours and maximal between 0.3 and 1.0 ng/ml of TGF-β. Higher concentrations of TGF-β were less stimulatory. The serum enhancement was dependent upon the simultaneous presence of TGF-β and serum factors and was abolished when chondrocytes were plated and exposed to TGF-β in medium containing dialyzed FBS (12-14 kD MW membrane). The nature of the uptake and incorporation of thymidine into DNA by individual chondrocytes appeared to be the same in both TGF-β-treated and control cultures as the apparent Ks were similar. The present study indicates that TGF-β increases DNA synthesis by growth plate chondrocytes. The effect is enhanced by factors present in serum. [ABSTRACT FROM AUTHOR]

Subcutaneous transplantation of growth plate chondrocytes isolated enzymatically from the proximal tibia of 6-week-old rabbits into athymic (nu/nu) mice resulted in the formation of cartilaginous nodules. Calcification of the matrix was first seen after 48 hrs, and endochondral ossification at 12 days. The mineral first occurred about hypertrophic cells. Histochemical alkaline phosphatase activity was concentrated in pericellular collars at the same location. Immunofluorescence examination with rabbit anti-mouse lymphocyte serum disclosed that the bulk of the osteoblasts was derived from the mouse. A small quantity of mouse antigen was present in the cartilage matrix at its junction with bone. It presumably diffused into the cartilaginous interface from the host, but the possibility that some chondrocytes were of murine origin has not been excluded. Five of six grafts of cells grown to confluence in monolayer culture for 10 to 14 days became ossified. The ability to induce mineralization declined in subculture. Chondrocytes killed by heating to 56 degrees did not induce calcified cartilage or bone. [ABSTRACT FROM AUTHOR]

A serum-free medium for postembryonic chicken epiphyseal growth plate chondrocytes has been developed from 104 MCDB medium. To enable these fastidious cells to survive, grow, and express normal phenotype, a substantial increase over MCDB 104 in the level of many of the amino acids was required, as well as a change in the buffer system and the addition of SerXtend, a defined, serum-free product containing various growth factors, including fibroblast growth factor. Also required was the provision of cell attachment factors, either by coating culture surfaces with type II collagen, or better, by allowing the freshly released cells to recover for several hours in a medium supplemented with 10% fetal bovine serum before plating. Ths new serum-free medium, which we call USC-HC1, supports growth and replication, the retention of normal polygonal morphology, the expression of significant levels of cellular alkaline phosphatase activity, the production of sulfated proteoglycans, type II collagen, and the formation of alkaline phosphatase-rich matrix vesicles by the chondrocytes. The major advantage of USC-HC1, however, is that it will provide for the first time an opportunity to examine the effects of various defined growth and hormonal factors on the phenotypic expression and differentiation of growth plate chondrocytes, in the absence of the variable (stimulatory and inhibitory) factors present in fetal bovine serum. [ABSTRACT FROM AUTHOR]

The effects of X-ray irradiation on the proliferation and differentiation of growth plate chondrocytes were examined. Chondrocytes were isolated from the ribs of 4-week-old New Zealand white rabbits and cultured as a packed mass in centrifuge tubes. The cells in the centrifuge tubes proliferated, matured and hypertrophied like in vivo chondrocytes. Irradiation at a dose as low as 3 Gy to proliferating chondrocytes significantly decreased the DNA content, alkaline phosphatase (ALPase) activity, and matrix calcification, without decreasing the proteoglycan content. Irradiation at 10Gy to mature chondrocytes caused significant decrease in ALPase activity and matrix calcification, although it did not have marked effects on DNA and proteoglycan contents. On the other hand, irradiation at 10Gy to hypertrophic chondrocytes did not cause any change in ALPase activity or cartilage-matrix calcification. These findings suggest that the irradiation sensitivity of chondrocytes changes during cytodifferentiation. Furthermore, the inhibition of ALPase expression and matrix calcification by irradiation proved to be independent of the suppression of proliferation. [ABSTRACT FROM AUTHOR]

Activating transcription factor 4 (Atf4) is a leucine-zipper-containing protein of the cAMP response element-binding protein (CREB) family. Ablation of Atf4 (Atf4-/-) in mice leads to severe skeletal defects, including delayed ossification and low bone mass, short stature and short limbs. Atf4 is expressed in proliferative and prehypertrophic growth plate chondrocytes, suggesting an autonomous function of Atf4 in chondrocytes during endochondral ossification. In Atf4-/- growth plate, the typical columnar structure of proliferative chondrocytes is disturbed. The proliferative zone is shortened, whereas the hypertrophic zone is transiently expanded. The expression of Indian hedgehog (Ihh) is markedly decreased, whereas the expression of other chondrocyte marker genes, such as type II collagen (Col2a1), PTH/PTHrP receptor (Pth1r) and type X collagen (Col10a1), is normal. Furthermore, forced expression of Atf4 in chondrocytes induces endogenous Ihh mRNA, and Atf4 directly binds to the Ihh promoter and activates its transcription. Supporting these findings, reactivation of Hh signaling pharmacologically in mouse limb explants corrects the Atf4-/- chondrocyte proliferation and short limb phenotypes. This study thus identifies Atf4 as a novel transcriptional activator of Ihh in chondrocytes that paces longitudinal bone growth by controlling growth plate chondrocyte proliferation and differentiation. [ABSTRACT FROM AUTHOR]