Discovery of sonic hedgehog expression in postnatal growth plate chondrocytes: differential regulation of sonic and Indian hedgehog by retinoic acid

Sonic hedgehog (Shh) is a key signal protein in early embryological patterning of limb bud development. Its analog, Indian hedgehog (Ihh), primarily expressed during early cartilage development in prehypertrophic chondrocytes, regulates proliferation and suppresses terminal differentiation of postnatal growth plate (GP) chondrocytes. We report here for the first time that both Shh and Ihh mRNA are expressed in the GP of rapidly growing 6-week-old broiler-strain chickens. They are also expressed in other tissues such as articular chondrocytes, kidney, and bone. In situ hybridization and RT-PCR analyses reveal Shh in all zones of the GP, with peak expression in late hypertrophy. Using primary cultures of GP chondrocytes in serum-containing medium, we followed the patterns of Shh and Ihh mRNA expression as the cultures matured and mineralized. We find a cyclical expression of both hedgehog genes during the early period of culture development between day 10 and 14; when one is elevated, the other tended to be suppressed, suggesting that the two hedgehogs may play complementary roles during GP development. Retinoic acid (RA), a powerful modulator of gene expression in cell differentiation, stimulates GP chondrocytes toward terminal differentiation, enhancing mineral formation. We find that RA strongly suppresses Ihh, but enhances expression of Shh in this system. While Ihh suppresses maturation of GP chondrocytes to hypertrophy, we hypothesize that Shh acts to push these cells toward hypertrophy.     

Smad7 regulates terminal maturation of chondrocytes in the growth plate

Members of the bone morphogenetic protein (BMP) superfamily, including transforming growth factor-betas (TGFβ), regulate multiple aspects of chondrogenesis. Smad7 is an intracellular inhibitor of BMP and TGFβ signaling. Studies in which Smad7 was overexpressed in chondrocytes demonstrated that Smad7 can impact chondrogenesis by inhibiting BMP signaling. However, whether Smad7 is actually required for endochondral ossification in vivo is unclear. Moreover, whether Smad7 regulates TGFβ in addition to BMP signaling in developing cartilage is unknown. In this study, we found that Smad7 is required for both axial and appendicular skeletal development. Loss of Smad7 led to impairment of the cell cycle in chondrocytes and to defects in terminal maturation. This phenotype was attributed to upregulation of both BMP and TGFβ signaling in Smad7 mutant growth plates. Moreover, Smad7−/− mice develop hypocellular cores in the medial growth plates, associated with elevated HIF1α levels, cell death, and intracellular retention of types II and X collagen. Thus, Smad7 may be required to mediate cell stress responses in the growth plate during development.     

PTHrP and Indian hedgehog control differentiation of growth plate chondrocytes at multiple steps

In developing murine growth plates, chondrocytes near the articular surface (periarticular chondrocytes) proliferate, differentiate into flat column-forming proliferating cells (columnar chondrocytes), stop dividing and finally differentiate into hypertrophic cells. Indian hedgehog (Ihh), which is predominantly expressed in prehypertrophic cells, stimulates expression of parathyroid hormone (PTH)-related peptide (PTHrP) which negatively regulates terminal chondrocyte differentiation through the PTH/PTHrP receptor (PPR). However, the roles of PTHrP and Ihh in regulating earlier steps in chondrocyte differentiation are unclear. We present novel mouse models with PPR abnormalities that help clarify these roles. In mice with chondrocyte-specific PPR ablation and mice with reduced PPR expression, chondrocyte differentiation was accelerated not only at the terminal step but also at an earlier step: periarticular to columnar differentiation. In these models, upregulation of Ihh action in the periarticular region was also observed. In the third model in which the PPR was disrupted in about 30% of columnar chondrocytes, Ihh action in the periarticular chondrocytes was upregulated because of ectopically differentiated hypertrophic chondrocytes that had lost PPR. Acceleration of periarticular to columnar differentiation was also noted in this mouse, while most of periarticular chondrocytes retained PPR signaling. These data suggest that Ihh positively controls differentiation of periarticular chondrocytes independently of PTHrP. Thus, chondrocyte differentiation is controlled at multiple steps by PTHrP and Ihh through the mutual regulation of their activities.     

Conditional Kif3a ablation causes abnormal hedgehog signaling topography, growth plate dysfunction, and excessive bone and cartilage formation during mouse skeletogenesis

The motor protein Kif3a and primary cilia regulate important developmental processes, but their roles in skeletogenesis remain ill-defined. Here we created mice deficient in Kif3a in cartilage and focused on the cranial base and synchondroses. Kif3a deficiency caused cranial base growth retardation and dysmorphogenesis, which were evident in neonatal animals by anatomical and micro-computed tomography (microCT) inspection. Kif3a deficiency also changed synchondrosis growth plate organization and function, and the severity of these changes increased over time. By postnatal day (P)7, mutant growth plates lacked typical zones of chondrocyte proliferation and hypertrophy, and were instead composed of chondrocytes with an unusual phenotype characterized by strong collagen II (Col2a1) gene expression but barely detectable expression of Indian hedgehog (Ihh), collagen X (Col10a1), Vegf (Vegfa), MMP-13 (Mmp13) and osterix (Sp7). Concurrently, unexpected developmental events occurred in perichondrial tissues, including excessive intramembranous ossification all along the perichondrial border and the formation of ectopic cartilage masses. Looking for possible culprits for these latter processes, we analyzed hedgehog signalling topography and intensity by monitoring the expression of the hedgehog effectors Patched 1 and Gli1, and of the hedgehog-binding cell-surface component syndecan 3. Compared with controls, hedgehog signaling was quite feeble within mutant growth plates as early as P0, but was actually higher and was widespread all along mutant perichondrial tissues. Lastly, we studied postnatal mice deficient in Ihh in cartilage; their cranial base defects only minimally resembled those in Kif3a-deficient mice. In summary, Kif3a and primary cilia make unique contributions to cranial base development and synchondrosis growth plate function. Their deficiency causes abnormal topography of hedgehog signaling, growth plate dysfunction, and un-physiologic responses and processes in perichondrial tissues, including ectopic cartilage formation and excessive intramembranous ossification.     

Nmyc upregulation by sonic hedgehog signaling promotes proliferation in developing cerebellar granule neuron precursors

Hedgehog pathway activation is required for expansion of specific neuronal precursor populations during development and is etiologic in the human cerebellar tumor, medulloblastoma. We report that sonic hedgehog (Shh) signaling upregulates expression of the proto-oncogene Nmyc in cultured cerebellar granule neuron precursors (CGNPs) in the absence of new protein synthesis. The temporal-spatial expression pattern of Nmyc, but not other Myc family members, precisely coincides with regions of hedgehog proliferative activity in the developing cerebellum and is observed in medulloblastomas of Patched (Ptch) heterozygous mice. Overexpression of Nmyc promotes cell-autonomous G(1) cyclin upregulation and CGNP proliferation independent of Shh signaling. Furthermore, Myc antagonism in vitro significantly decreases proliferative effects of Shh in cultured CGNPs. Together, these findings identify Nmyc as a direct target of the Shh pathway that functions to regulate cell cycle progression in cerebellar granule neuron precursors.     

Aberrant death in dark chondrocytes of the avian growth plate

Growth plate chondrocytes of embryonic chick femurs were examined by electron microscopy, cytophotometry and autoradiography. Apart from the well-described 'light' chondrocyte, a different 'dark' type of chondrocyte was present, comprising 10 - 35% of the cell population. They were found at all stages of chondrocyte differentiation and in all ages of the femurs studied. Well developed rough endoplasmatic reticulum and Golgi complex, many secretory vesicles, energetically active mitochondria and a lot of glycogen, indicating high activity of the cytoplasm, were combined with low RNA synthesis, gentle margination and scattered compaction of the chromatin. DNA cytometry revealed that most of dark cells were diploid, but 15 - 30% were tetraploid, with the absence of an S-phase. Substantial loss of DNA was found in about 10% of dark chondrocytes. The TUNEL reaction demonstrated a limited number of DNA strand breaks. Advanced dark cells possessed the nuclear features of both apoptosis and necrosis. Besides chromomeric-chromonemic compaction, a chromatin arrangement similar to that of prometaphase and metaphase, as well as amitotic nuclear segregation, all of them degenerative, were found. Our interpretation is that the dark chondrocytes undergo an aberrant type of cell death which may be combined with aberrant cell cycle. Cell death of dark chondrocytes is preceded by a pre-mortal burst of secretion.     

Forkhead‐box R2 promotes metastasis and growth by stimulating angiogenesis and activating hedgehog signaling pathway in ovarian cancer

Overexpression of forkhead‐box R2 (FoxR2) is related to metastasis and progression of tumor. However, its biological functions in ovarian cancer (OC) progression remain unclear. Herein, we aimed to explore the changes in biological functions and molecular events related to FoxR2 overexpression. We found that FoxR2 is upregulated frequently in OC where these events are associated with worse histologic grade and poor survival. Enhanced expression of FoxR2 was related to cell growth, migration, and epithelial‐mesenchymal transition, whereas silencing of FoxR2 suppressed these malignant phenotypes. In addition, angiogenesis was stimulated by FoxR2 overexpression by enhancing vascular endothelial growth factor expression. Further mechanistic investigations revealed that the increase in cell surface FoxR2 promoted sonic hedgehog binding and signaling. Inhibiting hedgehog pathway with sonidegib decreased FoxR2‐induced migration and lung metastasis of OC cells, establishing the critical role of hedgehog signaling in mediating the effects of FoxR2 expression. Taken together, our results indicate that FoxR2 overexpression in OC contributes to malignant behavior in cancer cells, at least in part through stimulating angiogenesis and activation of the hedgehog signaling pathway. Hedgehog signaling pathway activation may be the key in tumor progression mediated by FoxR2.     

Downregulation of Wnt signaling by sonic hedgehog activation promotes repopulation of human tumor cell lines

Tumor repopulation after radiotherapy is a big obstacle for clinical cancer therapy. The molecular mechanisms of tumor cell repopulation after radiotherapy remain unclear. This study investigated the role of sonic hedgehog (SHH) and Wnt signaling pathways in tumor repopulation after radiotherapy in an in vitro repopulation model. In this model, irradiated dying tumor cells functioned as feeder cells, whereas luciferase-labeled living tumor cells acted as reporter cells. Proliferation of reporter cells was measured by bioluminescence imaging. Results showed that irradiated dying HT29 and Panc1 tumor cells significantly stimulated the repopulation of living cells in their respective cultures. In HT29 and Panc1 cells, radiation significantly inhibited Wnt activity. In the irradiated dying HT29 and Panc1 cells, the level of the activated nuclear β-catenin was significantly decreased. Treatment with the Wnt agonist 68166 significantly decreased, whereas treatment with Wnt antagonist significantly increased, repopulation in HT29 and Panc1 tumor cells in a dose-dependent manner. β-catenin short-hairpin RNA (shRNA) also significantly promoted tumor cell repopulation. The level of secreted frizzled related protein-1 (SFRP1), hedgehog and Gli1 were increased in irradiated cells. Our results highlight the interaction between Wnt and SHH signaling pathways in dying tumor cells and suggest that downregulation of Wnt signaling after SHH activation is negatively associated with tumor repopulation.KEY WORDS: Colon cancer, Pancreatic cancer, Radiotherapy, Repopulation, Wnt signaling     

Treatment of growth plate injury with microencapsulated chondrocytes

Background

Tissue-engineered cartilage has provided a promising method in the treatment of physeal growth arrest. This study was designed to investigate transplantation of microencapsulated allogeneic chondrocytes to treat the injured growth plate.

Methods

Allogeneic chondrocytes were encapsulated within alginate-polylysinealginate semipermeable membranes. Microencapsulated chondrocytes co-cultured with Bone Mesenchymal Stem Cells (BMSCs) were evaluated whether it could promote chondrogenesis of BMSCs. An experiment model of an injured growth plate was made by resecting the lateral half of the right distal femur physis in rabbits. Microencapsulated allogeneic chondrocytes, allogeneic chondrocytes as well as empty microcapsules were grafted into growth plate defects of 6-week-old rabbits. Histological and radiographic examinations were examined after transplantation up to 16 weeks.

Results

The histological study showed that BMSCs co-cultured with microencapsulated chondrocytes could produce GAG and II collagen similarly with chondrocytes. Angular deformity and length discrepancy in the group with microencapsulated allogeneic chondrocytes were less than those in other groups (p < 0.001). The histological study confirmed the viability of microencapsulated chondrocytes at 16 weeks postoperatively. The neogenetic chondrocytes of columnar arrangement have been found in the growth plate defect to prevent early ossification and closure of the growth plate.

Conclusions

The histological study confirmed the viability of microencapsulated chondrocytes at 16 weeks postoperatively. The neogenetic chondrocytes of columnar arrangement have been found in the growth plate defect to prevent early ossification and closure of the growth plate.     

Effects of leptin to cultured growth plate chondrocytes

OBJECTIVE: This study aimed to evaluate whether leptin has any effect on growth plate chondrocytes. METHODS: We studied the effects of exogenous leptin on cultured rabbit growth plate chondrocytes. This involved assessing [3H]thymidine incorporation, alkaline phosphatase (ALP) activity, proteoglycan production, leptin receptor (Ob-R) activity, and detection of Ob-R using Western blot analysis. RESULTS: The existence of Ob-R in growth plate chondrocytes was revealed by Western blot and Ob-R activity. Prior to semiconfluence, leptin increased [3H]thymidine incorporation while at the semiconfluent and early confluent stages, leptin promoted ALP activity and tended to promote proteoglycan production. CONCLUSION: Growth plate chondrocytes possess Ob-Rs, and leptin enhance chondrocyte proliferation and subsequent cell differentiation.     

Cyclooxygenases and prostaglandin E2 receptors in growth plate chondrocytes in vitro and in situ--prostaglandin E2 dependent proliferation of growth plate chondrocytes

Prostaglandin E2 (PGE2) plays an important role in bone development and metabolism. To interfere therapeutically in the PGE2 pathway, however, knowledge about the involved enzymes (cyclooxygenases) and receptors (PGE2 receptors) is essential. We therefore examined the production of PGE2 in cultured growth plate chondrocytes in vitro and the effects of exogenously added PGE2 on cell proliferation. Furthermore, we analysed the expression and spatial distribution of cyclooxygenase (COX)-1 and COX-2 and PGE2 receptor types EP1, EP2, EP3 and EP4 in the growth plate in situ and in vitro. PGE2 synthesis was determined by mass spectrometry, cell proliferation by DNA [3H]-thymidine incorporation, mRNA expression of cyclooxygenases and EP receptors by RT-PCR on cultured cells and in homogenized growth plates. To determine cellular expression, frozen sections of rat tibial growth plate and primary chondrocyte cultures were stained using immunohistochemistry with polyclonal antibodies directed towards COX-1, COX-2, EP1, EP2, EP3, and EP4. Cultured growth plate chondrocytes transiently secreted PGE2 into the culture medium. Although both enzymes were expressed in chondrocytes in vitro and in vivo, it appears that mainly COX-2 contributed to PGE2-dependent proliferation. Exogenously added PGE2 stimulated DNA synthesis in a dose-dependent fashion and gave a bell-shaped curve with a maximum at 10-8 M. The EP1/EP3 specific agonist sulprostone and the EP1-selective agonist ONO-D1-004 increased DNA synthesis. The effect of PGE2 was suppressed by ONO-8711. The expression of EP1, EP2, EP3, and EP4 receptors in situ and in vitro was observed; EP2 was homogenously expressed in all zones of the growth plate in situ, whereas EP1 expression was inhomogenous, with spared cells in the reserve zone. In cultured cells these four receptors were expressed in a subset of cells only. The most intense staining for the EP1 receptor was found in polygonal cells surrounded by matrix. Expression of receptor protein for EP3 and EP4 was observed also in rat growth plates. In cultured chrondrocytes, however, only weak expression of EP3 and EP4 receptor was detected. We suggest that in growth plate chondrocytes, COX-2 is responsible for PGE2 release, which stimulates cell proliferation via the EP1 receptor.     

Cytosolic free calcium concentrations in avian growth plate chondrocytes

Isolated avian growth plate chondrocytes convert the acetoxymethyl ester (AM) form of Fura-2 quickly and efficiently to the Ca2(+)-sensitive pentacarboxylic acid (FA) form. Control experiments indicate that the Kd for intracellular Fura-2/FA is very close to that of extracellular Fura-2/FA at the same ionic strength and pH and that the Fura-2/FA fluorescence from indicator converted by intracellular organelles is quite small. Correcting for the effects of extracellular Fura-2/FA and partial hydrolysis products has improved the accuracy of determination of intracellular [Ca2+] over earlier measurements in chondrocytes. Cytosolic [Ca2+] in isolated growth plate chondrocytes (containing cells from each maturational stage) is found to require approximately 9 hours to recover from the isolation process. After this recovery period, cytosolic [Ca2+] in these cells converges to approximately 70 nM regardless of the [Ca2+] of the recovery medium, suggesting regulation of cytosolic [Ca2+] to a set point. Chondrocytes that are separated into maturationally distinct fractions using countercurrent centrifugal elutriation show an increase in cytosolic [Ca2+] with cellular maturation. The least mature resting cells have a [Ca2+] near 57 nM, while the most mature hypertrophic cells are around 95 nM.     

Hyaluronidase expression in cultured growth plate chondrocytes during differentiation

Hyaluronan (HA) is a major component of the extracellular matrix of cartilage, contributes to its structural and functional integrity, and has various important roles in the differentiation of chondrocytes. HA metabolism is regulated by both anabolic and catabolic processes; however, the details have not yet been clarified. The purpose of this study was to clarify the expression patterns of hyaluronidase (HAase) mRNAs (from the relevant HAase genes: the HYALs) and HAase activity during chondrocyte differentiation. Cartilage tissue and growth plate chondrocytes were isolated from the ribs of 4-week-old male Japanese rabbits. The expression of HYAL mRNAs in cartilage was analyzed by in situ hybridization. The expression levels of HYAL mRNAs in the culture were analyzed for each of the chondrocyte differentiation stages by means of quantitative real-time polymerase chain reaction analysis. Enzymatic activity in the conditioned medium from the cultures was examined by using HA zymography and an enzyme-linked immunosorbent-like assay. The expression levels of HYAL1 and HYAL2 mRNAs were enhanced about 2.8-fold and 3.2-fold at the maximum during the early matrix forming stage, respectively, and by about 3.2-fold and 2.0-fold at the maximum in the hypertrophic stage, respectively. HYAL3 mRNA was not detected throughout the experimental period. HAase activity was enhanced at the early matrix forming and hypertrophic stages. These results suggest that selective expression of HYALs is essential for extracellular HA metabolism during chondrocyte differentiation.This research was supported by Grants-in-Aid (no. 11557166) for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan     

Phagocytosis of dying chondrocytes by osteoclasts in the mouse growth plate as demonstrated by annexin-V labelling

Endochondral ossification in the epiphyseal growth plate of long bones is associated with programmed cell death (PCD) of a major portion of the chondrocytes. Here we tested the hypothesis that at the ossification front of the epiphyseal growth plate osteoclasts preferentially phagocytose chondrocytes that are undergoing PCD. We injected biotin-labelled annexin-V (anx-V-biotin, an early marker of PCD) intravenously in young adult mice. After 30 min of labelling, long bones were recovered and the tissue distribution examined of anx-V-biotin-labelled cells in the growth plate using ABC-peroxidase histochemistry. Positive staining for anx-V-biotin was detected in hypertrophic chondrocytes still present in closed lacunae at some distance from the ossification front. At the ossification front, chondrocyte lacunae were opened and close contacts were seen between tartrate-resistant acid phosphatase-positive osteoclasts and hypertrophic cartilage cells. Osteoclasts were significantly more frequently in contact with anx-V-biotin-labelled chondrocytes than with unlabelled chondrocytes. Osteoclasts also contained labelled and unlabelled phagocytic fragments within their cytoplasm. We conclude that in the growth plate osteoclasts preferentially phagocytose hypertrophic chondrocytes that are dying, suggesting these dying cells may signal osteoclasts for their removal.