Abstract
Bone sialoprotein (BSP) is a multifunctional extracellular matrix (ECM) protein present in bone and cementum. Global in vivo ablation of BSP leads to bone mineralization defects, lack of acellular cementum, and periodontal breakdown. BSP harbors three main functional domains: N-terminal collagen-binding domain, hydroxyapatite-nucleating domain, and C-terminal RGD integrin-binding signaling domain. How each of these domains contributes to BSP function(s) is not understood. We hypothesized that collagen-binding and RGD domains play distinct roles in cementoblast functions. Three CRISPR/Cas9 gene-edited cell lines were derived from control wild-type (WT) OCCM.30 murine immortalized cementoblasts: 1) deletion of the N-terminus of BSP after signal peptide, including entire collagen binding domain (Ibsp(∆N-Term)); 2) deletion of exon 4 (majority of collagen-binding domain; Ibsp(∆Ex4)); and 3) deletion of C-terminus of BSP including the integrin binding RGD domain (Ibsp(∆C-Term)). Compared to WT, Ibsp(∆Ex4) and Ibsp(∆C-Term) cell lines showed reduced BSP secretion, in vitro. Abnormal cell morphology was observed in all mutant cell lines, with Ibsp(∆C-Term) showing highly disorganized cytoskeleton. All mutant cell lines showed significantly lower cell proliferation compared to WT at all timepoints. Ibsp(∆N-Term) cells showed reduced cell migration by 24 h. All mutants exhibited over 50 % significant reduced mineralization at days 6 and 10. While WT cells were largely unaffected by seeding density, mutant cells failed to mineralize at lower cell density. Mutant cell lines diverged from WT and from each other by dysregulated expression in 23 genes involved in mineralization, ECM, and cell signaling. In summary, disabling BSP functional domains led to profound and distinct changes in cementoblast cell functions, especially dysregulated gene expression and reduced mineralization, in a way did not align with a straightforward narrative where each functional domain caused specific, expected differences. Instead, the study uncovered a significant level of complexity in how different mutant forms of BSP affected cell functions, in vitro.