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G-Protein-Coupled Estrogen Receptor-1 Positively Regulates the Growth Plate Chondrocyte Proliferation in Female Pubertal Mice.

ABSTRACT: Estrogen enhances long bone longitudinal growth during early puberty. Growth plate chondrocytes are the main cells that contribute to long bone elongation. The role of G-protein-coupled estrogen receptor-1 (GPER-1) in regulating growth plate chondrocyte function remains unclear. In the present study, we generated chondrocyte-specific GPER-1 knockout (CKO) mice to investigate the effect of GPER-1 in growth plate chondrocytes. In control mice, GPER-1 was highly expressed in the growth plates of 4- and 8-week-old mice, with a gradual decline through 12 to 16 weeks. In CKO mice, the GPER-1 expression in growth plate chondrocytes was significantly lower than that in the control mice (80% decrease). The CKO mice also showed a decrease in body length (crown-rump length), body weight, and the length of tibias and femurs at 8 weeks. More importantly, the cell number and thickness of the proliferative zone of the growth plate, as well as the thickness of primary spongiosa and length of metaphysis plus diaphysis in tibias of CKO mice, were significantly decreased compared with those of the control mice. Furthermore, there was also a considerable reduction in the number of proliferating cell nuclear antigens and Ki67-stained proliferating chondrocytes in the tibia growth plate in the CKO mice. The chondrocyte proliferation mediated by GPER-1 was further demonstrated via treatment with a GPER-1 antagonist in cultured epiphyseal cartilage. This study demonstrates that GPER-1 positively regulates chondrocyte proliferation at the growth plate during early puberty and contributes to the longitudinal growth of long bones.

SUBMITTER: Chou YS

PROVIDER: S-EPMC8417792 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:TGFβs act through canonical and non-canonical pathways, and canonical signals are transduced via Smad2 and Smad3. However, the contribution of canonical vs. non-canonical pathways in cartilage is unknown because the role of Smad2 in chondrogenesis has not been investigated in vivo. Therefore, we analyzed mice in which Smad2 is deleted in cartilage (Smad2CKO), global Smad3-/- mutants, and crosses of these strains. Growth plates at birth from all mutant strains exhibited expanded columnar and hypertrophic zones, linked to increased proliferation in resting chondrocytes. Defects were more severe in Smad2CKO and Smad2CKO;Smad3-/- (Smad2/3) mutant mice than in Smad3-/- mice, demonstrating that Smad2 plays a role in chondrogenesis. Increased levels of Ihh RNA, a key regulator of chondrocyte proliferation and differentiation, were seen in prehypertrophic chondrocytes in the three mutant strains at birth. In accordance, TGFβ treatment decreased Ihh RNA levels in primary chondrocytes from control (Smad2fx/fx) mice, but inhibition was impaired in cells from mutants. Consistent with the skeletal phenotype, the impact on TGFβ-mediated inhibition of Ihh RNA expression was more severe in Smad2CKO than in Smad3-/- cells. Putative Smad2/3 binding elements (SBEs) were identified in the proximal Ihh promoter. Mutagenesis demonstrated a role for three of them. ChIP analysis suggested that Smad2 and Smad3 have different affinities for these SBEs, and that the repressors SnoN and Ski were differentially recruited by Smad2 and Smad3, respectively. Furthermore, nuclear localization of the repressor Hdac4 was decreased in growth plates of Smad2CKO and double mutant mice. TGFβ induced association of Hdac4 with Smad2, but not with Smad3, on the Ihh promoter. Overall, these studies revealed that Smad2 plays an essential role in the development of the growth plate, that both Smads 2 and 3 inhibit Ihh expression in the neonatal growth plate, and suggested they accomplish this by binding to distinct SBEs, mediating assembly of distinct repressive complexes.

Project description:Loss of PTPN11/SHP2 in mice or in human metachondromatosis (MC) patients causes benign cartilage tumors on the bone surface (exostoses) and within bones (enchondromas). To elucidate the mechanisms underlying cartilage tumor formation, we investigated the role of SHP2 in the specification, maturation and organization of chondrocytes. Firstly, we studied chondrocyte maturation by performing RNA-seq on primary chondrocyte pellet cultures. We found that SHP2 depletion, or inhibition of the ERK1/2 pathway, delays the terminal differentiation of chondrocytes from the early-hypertrophic to the late-hypertrophic stage. Secondly, we studied chondrocyte maturation and organization in mice with a mosaic postnatal inactivation of Ptpn11 in chondrocytes. We found that the vertebral growth plates of these mice have expanded domains of early-hypertrophic chondrocytes that have not yet terminally differentiated, and their enchondroma-like lesions arise from chondrocytes displaced from the growth plate due to a disruption in the organization of maturation and ossification zones. Furthermore, we observed that lesions from human MC patients also display disorganized chondrocyte maturation zones. Next, we found that inactivation of Ptpn11 in Fsp1-Cre-expressing fibroblasts induces exostosis-like outgrowths, suggesting that loss of SHP2 in cells on the bone surface and at bone-ligament attachment sites induces ectopic chondrogenesis. Finally, we performed lineage tracing to show that exostoses and enchondromas in mice likely contain mixtures of wild-type and SHP2-deficient chondrocytes. Together, these data indicate that in patients with MC, who are heterozygous for inherited PTPN11 loss-of-function mutations, second-hit mutations in PTPN11 can induce enchondromas by disrupting the organization and delaying the terminal differentiation of growth plate chondrocytes, and can induce exostoses by causing ectopic chondrogenesis of cells on the bone surface. Furthermore, the data are consistent with paracrine signaling from SHP2-deficient cells causing SHP2-sufficient cells to be incorporated into the lesions.

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G-Protein-Coupled Estrogen Receptor-1 Positively Regulates the Growth Plate Chondrocyte Proliferation in Female Pubertal Mice.

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