Early insights into the role of Exoc6B associated with spondyloepimetaphyseal dysplasia with joint laxity type 3 in primary ciliogenesis and chondrogenic differentiation in vitro.

ABSTRACT

BACKGROUND: Spondyloepimetaphyseal dysplasia with joint laxity type 3 (SEMDJL3) is a rare skeletal dysplasia associated with EXOC6B, a component of the exocyst complex, involved in vesicle tethering and exocytosis at the plasma membrane. So far, EXOC6B and the pathomechanisms underlying SEMDJL3 remain obscure. METHODS AND RESULTS: Exoc6b was detected largely at the perinuclear regions and the primary cilia base in ATDC5 prechondrocytes. Its shRNA lentiviral knockdown impeded primary ciliogenesis. In Exoc6b silenced prechondrocytes, Hedgehog signaling was attenuated, including when stimulated with Smoothened agonist. Exoc6b knockdown deregulated the mRNA and protein levels of Col2a1, a marker of chondrocyte proliferation at 7- and 14-days following differentiation. It led to the upregulation of Ihh another marker of proliferative chondrocytes. The levels of Col10a1, a marker of chondrocyte hypertrophy was enhanced at 14 days of differentiation. Congruently, Axin2, a canonical Wnt pathway modulator that inhibits chondrocyte hypertrophy was repressed. The expression of Mmp13 and Adamts4 that are terminal chondrocyte hypertrophy markers involved in extracellular matrix (ECM) remodelling were downregulated at 7 and 14 days of chondrogenesis. Bglap that encodes for the most abundant non-collagenous bone matrix constituent and promotes ECM calcification was suppressed at 14 days of chondrocyte differentiation. ECM mineralization was assessed by Alizarin Red staining. Gene expression and ciliogenesis were investigated by reverse transcription quantitative real-time PCR, immunoblotting, and immunocytochemistry. CONCLUSIONS: These findings provide initial insights into the potential role of Exoc6b in primary ciliogenesis and chondrogenic differentiation, contributing towards a preliminary understanding of the molecular pathomechanisms underlying SEMDJL3.