The present study revealed that Angptl1 overexpression significantly inhibited number of TRAP-positive MNCs as well as the mRNA levels of TRAP and Ctsk in RAW 264.7 cells, indicating that Angptl1 suppresses osteoclast formation, then might lead to a suppression of bone resorption. The mechanisms by which Angptl1 suppresses osteoclast formation have still remained unknown at the present time. Since the effects of Angptl1 on NFATc1 expression seemed to be less than on those of TRAP and Ctsk in RAW 264.7 cells, Angptl1 might differently affect several signaling for osteoclast formation in mice. It has previously been reported that Angptl1 exerts anti-apoptotic activity through the phosphorylation of ERK1/2 and Akt in zebrafish endothelial cells . Moreover, the studies of Akt-deficient mice suggested that Akt signaling is partly related to osteoblastic bone formation and osteoclastic bone resorption in bone remodeling [15, 16], although the previous studies suggest that ERK1/2 and Akt signaling stimulates bone resorption in mice in contrast to the effects of Angptl1 on osteoclast formation .
Lu et al. reported that Angptl7, an Angptl family member protein, induces BMP expression and enhances the proliferation and differentiation through the BMP autocrine loop in mouse osteoblastic MC3T3-E1 cells . Angptl2 has been shown as a positive regulator of osteoblastic differentiation, and its expression was suppressed by inflammatory stimuli in vitro . Meanwhile, Angptl2 enhanced cementoblast differentiation partially by activating the ERK1/2 signaling pathway in mouse cementoblast cell line . However, there has been no evidence of Angptl1 in bone metabolism so far. In the present study, Angptl1 overexpression significantly enhanced Osterix mRNA levels, ALP activity and mineralization in the presence of BMP-2 in mouse mesenchymal ST2 cells, although it did not affect the mRNA levels of Runx2, Osterix, ALP and osteocalcin in mouse osteoblastic MC3T3-E1 cells. These data suggest that Angptl1 enhances the mesenchymal cells into osteoblasts at the early stage of osteogenic differentiation, then leading to an enhancement of ALP activity and mineralization. In the present study, Angptl1 overexpression significantly suppressed osteocalcin mRNA levels enhanced by BMP-2 in ST2 cells, suggesting that Angptl1 affects osteoblastic differentiation at the early stage, but not the terminal stage, since osteocalcin has been considered to be late stage osteoblast differentiation marker .
Angptl2 has been well-studied in glucose/lipid metabolism. Kadomatsu et al. demonstrated that Angptl2 possesses angiogenic effects, which are related to the chronic inflammation in obesity and cancer metastasis [22–24]. Serum levels of Angptl2 have also been reported in type 2 diabetic and obese patients [25, 26]. These in vitro and clinical studies suggested that Angptl2 is involved in the pathophysiology of metabolic syndrome, such as obesity, although no reports are available about the roles of Angptl1 in adipobiology. We therefore examined the effects of Angptl1 on adipogenic differentiation, since bone metabolism is regulated by the trans differentiation of mesenchymal stem cells into osteogenic or adipogenic cells . In the present study, Angptl1 overexpression significantly decreased the mRNA levels of PPARγ and aP2 during adipogenic induction in mouse preadipocyte cell line, 3T3-L1 cells, suggesting that Angptl1 suppresses adipogenic differentiation in mouse cells. Taken into account into the enhancement of Angptl1 on the mesenchymal cells into osteoblasts at the early stage, Angptl1 induces trans differentiation from adipogenic-lineage into osteogenic lineage cells during bone remodeling or in the pathological state, such as diabetes and obesity.
Bone modeling and remodeling process participate during bone repair after fractures or bone defect . Chondrogenesis, bone formation and bone resorption as well as vessel formation and inflammation are related to this bone repair process. Tanoue et al. evaluated Angptl2 expression and function in chondrocyte differentiation and subsequent endochondral ossification in mice, and they showed that Angptl2 functions as an extracellular matrix protein in chondrocyte differentiation and subsequent endochondral ossification . Therefore, Angptl1 might participate bone repair process by affecting chondrogenesis, bone restoration and remodeling phase, our preliminary study showed that an elevation in Angptl1 mRNA level are not evident at the injured sites after femoral bone defect in mice (data not shown). The roles of Angptl1 in chondrogenesis and bone repair have still remained unknown.