Of 953 transcripts with differential abundance in OCPs from SHP2Prrx1KO;R26mTmG and SHP2Prrx1CTR;R26mTmG mice, 397 increased, and 556 decreased. the PKA signaling pathway. Our data BMS-708163 (Avagacestat) indicate that SHP2 is critical for skeletal cell lineage differentiation and could thus be a pharmacologic target for bone and cartilage regeneration. Introduction Vertebrate skeletal development occurs through intramembranous and endochondral ossification. Intramembranous ossification involves the direct differentiation of mesenchymal stem cells into osteoblasts and is responsible for the ossification of cranial bones and for appositional bone BMS-708163 (Avagacestat) growth.1,2 Endochondral ossification requires the formation of cartilaginous anlagen and their subsequent replacement by osteoblasts, and contributes to longitudinal bone growth.3,4 During endochondral ossification, mesenchymal cells condense and then differentiate into early proliferating chondrocytes, which undergo further differentiation to establish a cartilage growth plate. Cells within growth plates are organized Mouse monoclonal to TrkA into distinct zones containing resting, proliferating, pre-hypertrophic, and hypertrophic chondrocytes. Hypertrophic chondrocytes undergo apoptosis and are replaced by osteoblasts or transdifferentiate into osteoblasts, which produce bone.5C10 Signaling molecules and transcription factors, including SOX9,11,12 -CATENIN,13 and RUNX2,14,15 regulate skeletal development. The transcription factor SOX9 is a master regulator of chondrogenesis, essential for chondrocyte specification, proliferation, and early differentiation.12,16,17 SOX9 promotes the expression of important chondrocytic genes, including cause Noonan and LEOPARD syndromes (NS and LS, respectively), which feature skeletal manifestations that can include pectus carinatum or pectus excavatum, short stature, and scoliosis.33,34 Heterozygous SHP2 loss-of-function (LOF) mutations are responsible for the autosomal dominant disorder metachondromatosis, in which somatic second hit mutations give rise to enchondromas and exostoses.35,36 We and others have demonstrated that inactivation of in cells committed to the chondrogenic lineage impairs terminal differentiation to chondrocytes, and inactivation at other sites may promote chondrogenesis instead of osteogenesis.37,38 However, the role of SHP2 in modulating cell fate decisions in OCPs remains unexplored. By utilizing a tissue-specific gene ablation approach, we report here that SHP2 deficiency in both limb and head mesenchymal progenitors impairs cartilage, bone and joint development. SHP2 regulates chondrogenesis by modulating the lineage commitment of mesenchymal progenitors and by repressing chondrocytic differentiation, and this regulation is mediated at least in part by influencing the phosphorylation and SUMOylation of SOX9 via the PKA signaling pathway. Results SHP2 deficiency in limb and head mesenchyme affects skeletogenesis To investigate the role of SHP2 in limb and head mesenchymal cells during early skeletogenesis, mice carrying floxed ((SHP2Prrx1CTR), (SHP2Prrx1KO), (SHP2Prrx1CTR/ER) and (SHP2Prrx1KO/ER) mice (Fig.?S1a). The promoter is active both in the undifferentiated mesenchyme of limb buds42 and in the periosteum of adult mice.43 Therefore, in SHP2Prrx1KO and SHP2Prrx1KO/ER mice, is specifically deleted in PRRX1-expressing mesenchymal osteochondroprogenitors (OCPs) and their progeny. The deletion efficiency of floxed alleles in OCPs and their derivatives by or was determined by Western blot analysis, which revealed that SHP2 abundance was reduced by?>?80% and?>?70% in purified OCPs and their derivatives from SHP2Prrx1KO or tamoxifen-treated SHP2Prrx1KO/ER mice respectively, compared with those from SHP2Prrx1CTR and SHP2Prrx1CTR/ER controls (Fig.?S1b). SHP2Prrx1CTR and SHP2Prrx1CTR/ER mice had no discernible phenotype, so subsequent analyses were focused on SHP2Prrx1KO and SHP2Prrx1KO/ER mice. SHP2Prrx1KO mice were born at the expected Mendelian ratios and they were the same size as the SHP2Prrx1CTR littermate controls at birth, on average [(48.8??3.5)mm vs. (49.0??4.2)mm long at P0.5, floxed allele to is expressed in?committed osteoblasts, this deletion differentiates the roles for SHP2 in OCPs and fully differentiated osteoblastic cells. Importantly, mice had normal appearing trabecular and cortical bone at day P0.5 and by 8 weeks old (Fig.?S10), which was BMS-708163 (Avagacestat) not the case for mice. These results strongly suggest that SHP2’s major role occurs during OCP commitment to the osteoblast lineage. Open in a separate window Fig. 2 SHP2 deficiency in PRRX1-expressing OCPs delays endochondral ossification and leads to ectopic cartilage formation. a Representative images of H&E-stained longitudinal sections of femurs demonstrate impaired ossification BMS-708163 (Avagacestat) of appendicular bones, enhanced chondrogenesis and ectopic cartilage formation in 7-day-old SHP2Prrx1KO mice, compared with SHP2Prrx1CTR Mice. Bottom panels are enlarged views (10) of corresponding.