We employed a global gene expression profiling using DNA microarrays to characterise non-canonical osteogenic factors regulating the differentiation of bone marrow skeletal stem cells (marrow stromal stem cells, BMSCs) into osteoblastic cells. We identified collapsing response mediator protein-4 (CRMP4) that was the only member of CRMP1-5 family to be expressed by BMSCs. We found CRMP4, a cytosolic phosphoprotein that mediates Semaphorin-3A effects in neuronal differentiation to be expressed by proliferating chondrocytes and osteoblastic cells and its expression was detected in bone lining osteoblasts in postnatal and adult mouse bones. In vitro gain and loss of CRMP4 function in bone marrow stromal cell line ST2 revealed the inhibitory effect of CRMP4 on osteoblast differentiation. Consistently, mice lacking Crmp4 expression displayed significant increased bone mass by 40% compared with WT controls due to increased trabecular and cortical bone microarchitecture parameters as measured by micro-CT analysis. Histomorphometic analysis revealed significant increased osteoblast number/bone surface in Crmp4−/− bone with no effect on osteoclastic bone resorption parameters compared with WT controls. Mechanistic studies revealed that increased bone mass in Crmp4−/− mice was associated with upregulation of BMP2-induced osteogenesis in Crmp4−/− osteoblasts (OB) as evidenced by enhanced activation of canonical and non-canonical BMP2 signalling. Furthermore, Crmp4−/− OB exhibited enhanced activation of RhoA/focal adhesion kinase (FAK) signalling that led to cytoskeletal changes associated with increased rate of cell spreading as well as increased cell proliferation rate by increasing the percentage of Crmp4−/− OB in S/G2/M phases of the cell cycle compared with WT OB. The later effect was mediated via inhibiting p21Cip/Waf and upregulating cyclin D1 expression, two targets of RhoA pathway. These findings identify the neuro-protein CRMP4 as a novel negative regulator of bone formation by inhibiting BMP-induced osteogenesis and RhoA-stimulated OB proliferation. Thus, CRMP4 is a new therapeutic target for enhancing bone formation.
Disclosure: The authors declared no competing interests. The Danish Research council (DFF 4004-00045) and University of Southern Denmark (SDU 647-108).