Discussion
The present study provides evidence that a physiologic role of Sam68 is to modulate bone marrow mesenchymal stem cells. Young Sam68−/− mice developed normally and contained similar bone mass compared with wild-type littermates. Aged (12 months) Sam68−/− mice displayed a high bone mass phenotype compared with Sam68+/+ littermates. The wild-type littermates underwent age-related bone loss that occurs naturally in mammals, while the Sam68−/− animals preserved their bone mass with aging. The differentiation of bone marrow stem cells isolated from Sam68−/− mice and embryonic mesenchymal multipotential progenitor cells C3H10T1/2 treated with Sam68 shRNA resulted in a more pronounced osteoblast differentiation. These findings demonstrate that the loss of Sam68 enhances osteoblast differentiation. The converse was also true, as MEFs isolated from Sam68−/− animals were impaired in their ability to undergo adipocyte differentiation compared with Sam68+/+ MEFs. These findings suggest that a physiologic role for Sam68 is to regulate the balance between adipogenic and osteogenic differentiation of the bone marrow mesenchymal.
Sam68 protein expression was observed throughout the developing mouse embryo, in keeping with previous reports that identified the Sam68 mRNA to be widely expressed [38]. We observe Sam68 staining in the brain, heart, and intestine (see Figure 1) as well as liver, skin, and kidney (unpublished data) of late-stage embryos. This expression pattern is consistent with previous work that has observed Sam68 in neurons [52] and as a substrate of the intestinal SIK/breast tumor kinase [43]. The expression of Sam68 was not altered in aging bone marrow stromal cells or in senescencing WI-38 cells (unpublished data). The presence of Sam68 in chondrocytes, osteoblasts and osteoclasts in developing cartilage and bone predicted a pivotal role for the protein in skeletal development.
The Sam68−/− mice were generated using a traditional targeting approach where the functional KH domain was deleted and approximately one third of the Sam68−/− mice survived to adulthood with no apparent defects. One explanation for this phenomenon is that Sam68 function is sub-served by one or more of its family members during development such as SLM-1 and SLM-2 [47]. Alternatively, Sam68 is not required during embryonic development. However, two thirds of the Sam68−/−, but not the Sam68+/- pups, were killed by their Sam68+/- mothers. These findings suggest that the mothers are able to detect a subtle defect/difference that we cannot. Adult Sam68−/− mice live a normal life span of approximately two years. The Sam68−/− males were sterile and the Sam68−/− females provided inadequate care to their young, but the pups were not scattered and neglected as observed with FosB−/− mice [53]. Serum levels of estrogen decreased in aging Sam68−/− females as expected; however, the leptin levels decreased in aged Sam68−/− females. The aged Sam68−/− females were not obese and actually weighed less than the littermate controls (see Table 2). Moreover, their appetite was not altered with aging (N. Torabi and S. Richard, unpublished data), suggesting that the observed leptin reduction was not recreating ob/ob-like phenotypes related to weight, appetite and female sterility [54].
The skeletal phenotyping of cohorts of Sam68+/+ and Sam68−/− mice showed that bone mass was preserved in aged Sam68−/− mice. Traditional histology and histomorphometry suggested that the mechanism involved preservation of osteoblast and osteoclast activity. The documented role of the Sam68 regulatory protein, Src, in osteopetrosis led us to investigate the morphology and activity of Sam68−/− osteoclasts ex vivo. The Src tyrosine kinase was shown to play a role in bone remodeling when Src−/− mice died at 6 months of age with an osteopetrotic phenotype [48] and the defect was attributed to defective osteoclast function [55−59]. We therefore cultured mature osteoclasts harvested from Sam68+/+ and Sam68−/− mice ex vivo on dentin slices to quantify their resorptive capacity. The fact that Sam68−/− osteoclasts looked and acted like Sam68+/+ osteoclasts ex vivo and in vivo made it unlikely that this was the primary source of the difference in bone metabolism in 12-month-old Sam68−/− mice. The fact that the CTX levels were lower in young and old Sam68−/− mice suggested that there is reduced bone resorption compared with wild-type littermate controls. However, this reduction in bone resorption occurred with normal osteoclast activity, as assessed by in vitro culturing. These observations are consistent with the Sam68−/− mice having a youth-like bone phenotype. However, it is still possible, that a mild impairment in Sam68−/− mice osteoclast function may manifest itself later in life in overall accumulation of bone and this will require further detailed studies.
Ex vivo differentiation of primary bone marrow stromal cells, harvested from Sam68+/+ and Sam68−/− mice, down the osteoblast lineage revealed an osteogenic advantage in the cultures of cells derived from the Sam68−/− mice. It will be important to demonstrate that a similar effect is observed using primary bone marrow stromal cells from aged Sam68−/− mice. Similar findings were observed when embryonic mesenchymal multipotential progenitor cells C3H10T1/2 treated with Sam68 shRNA, were differentiated into osteoblasts with BMP2. Our findings that aged Sam68−/− mice do not develop fatty bone marrow and that MEFs derived from Sam68 −/− mice have impaired adipocyte differentiation, indicate that Sam68 regulates both adipocyte and osteoblast differentiation. These findings identify Sam68 as the first RNA binding protein to regulate mesenchymal cell differentiation and the challenge will be to identify the specific RNA targets that it regulates during this process. The fact that osteoblast function is altered in Src−/− mice [60,61] raises the possibility that preservation of bone mass in the Sam68−/− mice could be linked with and regulated by Src.
The pathway by which leptin regulates bone resorption was identified to involve the sympathetic nervous system relaying to the osteoblasts via the β-adrenergic pathway leading to the release of growth factors including RANKL that causes the osteoclasts to thrive [8,18–20]. The lowering of leptin levels in aged Sam68−/− mice is consistent with these mice having a high bone mass compared with their aged littermates. These data would suggest that the leptin-sympathetic pathway is unaltered in Sam68−/− mice and that the lowering of leptin may explain the lower levels of CTX in the serum of Sam68−/− mice. These findings suggest that Sam68 may be regulating bone metabolism at two different levels: (1) the absence of Sam68 results in lower leptin levels that may reduce bone resorption via the sympathetic nervous system and (2) the absence of Sam68 favors osteoblast, rather than adipocyte, differentiation.
In conclusion, our data define a physiologic role for Sam68 in bone metabolism and bone marrow mesenchymal stem cell differentiation. The bone phenotype observed in Sam68−/− mice imply that inhibitors of Sam68 could prevent age-related bone loss. Furthermore, the results also suggest that Sam68 expression levels, hypomorphism, and mutations in humans may influence susceptibility to marrow adipocyte accumulation and osteoporosis. Our findings also identify a new animal model to study aging bone loss.