Leptin Antagonizes Peroxisome Proliferator-Activated Receptor-γ Signaling in Growth Plate Chondrocytes.

Leptin is an obesity-associated cytokine-like hormone encoded by the ob gene. Recent studies reveal that leptin promotes proliferation and differentiation of chondrocytes, suggesting a peripheral role of leptin in regulating growth plate function. Peroxisome proliferator-activated receptor-γ (PPARγ) is a transcriptional regulator of adipogenesis. Locally, PPARγ negatively regulates chondrogenic differentiation and terminal differentiation in the growth plate. The aim of this study was to test the hypothesis that leptin may suppress the inhibitory effects of PPARγ on growth plate chondrocytes. Chondrocytes were collected from distal femoral growth plates of newborn rats and were cultured in monolayer or cell pellets in the presence or absence of leptin and the PPARγ agonist ciglitazone. The results show that leptin attenuates the suppressive effects of PPARγ on chondrogenic differentiation and T3-mediated chondrocyte hypertrophy. Leptin treatment also leads to a mild downregulation of PPAR mRNA expression and a significant MAPK/ERK-dependent PPARγ phosphorylation at serine 112/82. Blocking MAPK/ERK function with PD98059 confirmed that leptin antagonizes PPARγ function in growth plate chondrocytes through the MAPK/ERK signaling pathway. Furthermore, leptin signaling in growth plate cells is also negatively modulated by activation of PPARγ, implying that these two signaling pathways are mutually regulated in growth plate chondrocytes.

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.

Primary cilia modulate Ihh signal transduction in response to hydrostatic loading of growth plate chondrocytes.

Indian hedgehog (Ihh) is a key component of the regulatory apparatus governing chondrocyte proliferation and differentiation in the growth plate. Recent studies have demonstrated that the primary cilium is the site of Ihh signaling within the cell, and that primary cilia are essential for bone and cartilage formation. Primary cilia are also postulated to act as mechanosensory organelles that transduce mechanical forces acting on the cell into biological signals. In this study, we used a hydrostatic compression system to examine Ihh signal transduction under the influence of mechanical load. Our results demonstrate that hydrostatic compression increased both Ihh gene expression and Ihh-responsive Gli-luciferase activity. These increases were aborted by disrupting the primary cilia structure with chloral hydrate. These results suggest that growth plate chondrocytes respond to hydrostatic loading by increasing Ihh signaling, and that the primary cilium is required for this mechano-biological signal transduction to occur.

Leptin synergizes with thyroid hormone signaling in promoting growth plate chondrocyte proliferation and terminal differentiation in vitro.

Leptin and thyroid hormone are two hormones that regulate energy balance through central signaling mechanisms. Recent studies in leptin-deficient ob/ob mice indicate that leptin also has peripheral effects in modulating the function of the growth plate, perhaps in terms of proliferation and differentiation enhancement. Thyroid hormone has been well-described as a potent stimulator of growth plate chondrocyte maturation. The objective of this study was therefore to investigate the interaction between leptin and thyroid hormone signaling in growth plate chondrocyte proliferation and terminal differentiation. Our in vitro data demonstrate that leptin synergistically functions with thyroid hormone through activation of both IGF-1/IGF1R signaling and Wnt/ß-catenin signaling, two pathways that have been previously described as downstream effectors of thyroid hormone action. Leptin increases thyroid hormone receptor-α (TRα) expression and thyroid hormone receptor transcriptional activity. Thyroid hormone also activates leptin signaling in growth plate cells undergoing proliferation and hypertrophy. We conclude that leptin synergically interacts with thyroid hormone in promoting growth plate chondrocyte proliferation and terminal differentiation.

Thyroid hormone-mediated growth and differentiation of growth plate chondrocytes involves IGF-1 modulation of beta-catenin signaling.

Thyroid hormone regulates terminal differentiation of growth plate chondrocytes in part through modulation of the Wnt/beta-catenin signaling pathway. Insulin-like growth factor 1 (IGF-1) has been described as a stabilizer of beta-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/beta-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 beta-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. T(3) activates IGF-1/IGF1R signaling and IGF-1-dependent PI3K/Akt/GSK-3beta signaling in growth plate chondrocytes undergoing proliferation and differentiation to prehypertrophy. T(3)-mediated Wnt-4 expression, beta-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 beta-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/GSK3beta pathway, cell hypertrophy is likely due to activation of Wnt/beta-catenin signaling, which is at least in part initiated by IGF-1 signaling or the IGF-1-activated PI3K/Akt signaling pathway.

Carboxypeptidase Z (CPZ) links thyroid hormone and Wnt signaling pathways in growth plate chondrocytes.

Carboxypeptidase Z (CPZ) removes carboxyl-terminal basic amino acid residues, particularly arginine residues, from proteins. CPZ contains a cysteine-rich domain (CRD) similar to the CRD found in the frizzled family of Wnt receptors. We have previously shown that thyroid hormone regulates terminal differentiation of growth plate chondrocytes through activation of Wnt-4 expression and Wnt/beta-catenin signaling. The Wnt-4 protein contains a C-terminal arginine residue and binds to CPZ through the CRD. The objective of this study was to determine whether CPZ modulates Wnt/beta-catenin signaling and terminal differentiation of growth plate chondrocytes. Our results show that CPZ and Wnt-4 mRNA are co-expressed throughout growth plate cartilage. In primary pellet cultures of rat growth plate chondrocytes, thyroid hormone increases both Wnt-4 and CPZ expression, as well as CPZ enzymatic activity. Knockdown of either Wnt-4 or CPZ mRNA levels using an RNA interference technique or blocking CPZ enzymatic activity with the carboxypeptidase inhibitor GEMSA reduces the thyroid hormone effect on both alkaline phosphatase activity and Col10a1 mRNA expression. Adenoviral overexpression of CPZ activates Wnt/beta-catenin signaling and promotes the terminal differentiation of growth plate cells. Overexpression of CPZ in growth plate chondrocytes also removes the C-terminal arginine residue from a synthetic peptide consisting of the carboxyl-terminal 16 amino acids of the Wnt-4 protein. Removal of the C-terminal arginine residue of Wnt-4 by site-directed mutagenesis enhances the positive effect of Wnt-4 on terminal differentiation. These data indicate that thyroid hormone may regulate terminal differentiation of growth plate chondrocytes in part by modulating Wnt signaling pathways through the induction of CPZ and subsequent CPZ-enhanced activation of Wnt-4.

Thyroid hormone interacts with the Wnt/beta-catenin signaling pathway in the terminal differentiation of growth plate chondrocytes.

UNLABELLED: Thyroid hormone activates Wnt-4 expression and Wnt/beta-catenin signaling in rat growth plate chondrocytes. Wnt antagonists Frzb/sFRP3 and Dkk1 inhibit T3-induced Wnt/beta-catenin activation and inhibit the maturation-promoting effects of T3 in growth plate cells. This study indicates that thyroid hormone regulates terminal differentiation of growth plate chondrocytes in part through modulating Wnt/beta-catenin signaling. INTRODUCTION: Thyroid hormone is a potent regulator of skeletal maturation in the growth plate, yet the molecular mechanisms underlying this profound effect remain unknown. Wnt signaling has recently been recognized as an important signal transduction pathway in regulating chondrogenesis and terminal differentiation of growth plate chondrocytes. The objective of this study was to explore the interaction between the thyroid hormone and Wnt signaling pathways in the growth plate. MATERIALS AND METHODS: Rat epiphyseal chondrocytes were maintained in 3D pellet culture and treated with triiodothyronine (T3). Activation of Wnt/beta-catenin signaling pathway in response to T3 was detected by measurement of the expression of Wnt-4 mRNA, the cellular accumulation of beta-catenin, the transcriptional activity of TCF/LEF, and the expression of the Wnt/beta-catenin responsive gene Runx2/cbfa1. Terminal differentiation of the chondrocytes was assessed by measurement of alkaline phosphatase enzymatic activity and Col10a1 gene expression. RESULTS: Thyroid hormone treatment of growth plate chondrocytes upregulated both Wnt-4 mRNA and protein expression, increased cellular accumulation of stabilized beta-catenin, increased TCF/LEF transcriptional activity, and stimulated the expression of the Runx2/cbfa1 gene. Overexpression of either Wnt-4 or a stabilized form of beta-catenin promoted growth plate chondrocyte terminal differentiation. Blocking Wnt ligand/receptor interactions with the secreted Wnt antagonists Frzb/sFRP3 or Dkk1 inhibited these T3-induced increases in beta-catenin accumulation and Runx2 gene expression and inhibited the maturation-promoting effects of T3 in growth plate cells. CONCLUSIONS: These data suggest that thyroid hormone regulates terminal differentiation of growth plate chondrocytes in part through modulating canonical Wnt/beta-catenin signaling.

Analysis of gene expression in mineralized skeletal tissues by laser capture microdissection and RT-PCR.

The analysis of gene expression by growth plate chondrocytes in vivo has been hampered by the inherent difficulty in performing in situ hybridization on mineralized tissues. The combination of laser capture microdissection and reverse transcription-polymerase chain reaction (RT-PCR) allows analysis of gene expression by cells selectively removed from histologic sections by laser ablation. In order to apply this method to mineralized tissues, a decalcification process is required. The object of this study was to determine the optimal method for tissue decalcification prior to laser capture microdissection RT-PCR that will preserve integrity of the mRNA population. Acetone, 10% formalin, and methacarn were evaluated as fixatives, while Surgipath Decalicifier I, 10% ethylenediaminetetraacetic acid (EDTA), and 20% EDTA were evaluated as decalcifying reagents. Our results demonstrate that the optimal RNA quality was preserved by a decalcification protocol consisting of 20% EDTA for decalcification followed by fixation in methacarn, although this method is also associated with a reduction in RNA quantity.

Thyroid hormone and the growth plate.

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 thyroid 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.

Peroxisome Proliferator-Activated Receptor-gamma Promotes Adipogenic Changes in Growth Plate Chondrocytes In Vitro.

Chondrocytes and adipocytes are two differentiated cell types which are both derived from mesenchymal cells. The purpose of this study was to investigate whether peroxisome proliferator-activated receptor-gamma (PPARgamma), a transcription factor involved in lineage determination during adipogenesis, is able to induce adipogenic differentiation in growth plate chondrocytes. Isolated epiphyseal chondrocytes were infected with a PPARgamma adenovirus or treated with the PPARgamma agonist ciglitazone. Both of these treatments resulted in lipid droplet accumulation and expression of the adipogenic markers aP2, lipoprotein lipase, and adipsin in chondrocytes. Proteoglycan matrix synthesis was decreased in the PPARgamma-infected cells, as was the expression of the chondrogenic genes Col2a1 and aggrecan. Growth plate cells transfected with a PPARgamma expression plasmid under the control of the collagen alpha1(II) promoter also demonstrated a similar adipogenic changes. Terminal differentiation of growth plate chondrocytes induced by thyroid hormone was also inhibited by overexpression of PPARgamma and ciglitazone treatment, with decreased expression of alkaline phosphatase and Runx2/Cbfa1 genes. These in vitro data suggest that PPARgamma is able to promote adipogenic differentiation in growth plate chondrocytes, while negatively regulating chondrogenic differentiation and terminal differentiation.

Peroxisome proliferator activated receptor-gamma (PPARgamma) represses thyroid hormone signaling in growth plate chondrocytes.

Peroxisome proliferator activated receptors (PPARs) are DNA-binding nuclear hormone receptors that are upregulated in response to high fat diets. PPARs are structurally related to the type II nuclear receptors, including the thyroid hormone receptors (TRs). To investigate if PPARs modulate TR-mediated terminal differentiation of growth plate chondrocytes, primary cultures of epiphyseal chondrocytes transiently transfected with TRalpha and PPARgamma expression vectors were treated with the PPAR ligands ciglitazone or troglitazone. Forced overexpression of PPARgamma decreased TRalpha1-mediated transcriptional activity and suppressed T3-induced increases in alkaline phosphatase activity and type X collagen expression. Similar effects were observed when the cells were treated with the PPARgamma activator ciglitazone or troglitazone. Overexpression of retinoid X receptor-alpha (RXRalpha) partially restored not only the inhibition of transcriptional activation by PPARgamma but also T3-induced hypertrophic differentiation. These data demonstrate that activation of PPARgamma signaling by either addition of PPARgamma ligands or overexpression of PPARgamma in growth plate chondrocytes inhibits TR-mediated gene transcription and inhibits the biological effects of thyroid hormone on terminal differentiation. The molecular mechanism involved in this inhibition appears to be competition between PPARgamma and TRalpha for limiting amounts of the heterodimeric partner RXR.

Expression and activation of peroxisome proliferator-activated receptors in growth plate chondrocytes.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a nuclear hormone receptor that is involved in a wide range of cellular processes. Although it is known that PPAR-gamma plays an important role in cell cycle control, inflammation, apoptotic cell death, and other cellular processes, the role of PPAR-gamma in the normal and pathological function of growth plate chondrocytes has not been investigated. The purpose of this study was to determine if PPARs are expressed in growth plate chondrocytes and to describe the biological effect of PPAR activation in these cells. The results demonstrate the presence of three PPAR isoforms (alpha, delta, and gamma) in growth plate cartilage. Activation of PPAR-gamma by ciglitazone in growth plate chondrocytes inhibits T(3) induced terminal differentiation and promotes apoptosis through increased levels of caspase 3/7 activity and decreased expression of the anti-apoptotic protein Bcl-2.