In this study, VEGF was conditionally targeted in Osx-positive osteoblast lineage cells, allowing studies of the functions of osteoblast-derived VEGF. Our data demonstrate that osteoblast-derived VEGF stimulates formation of osteoblasts and suppresses adipogenesis, both in vivo and in vitro. Thus, normal postnatal bone homeostasis requires expression of VEGF in osteoblasts. The reduction in bone mass and the increase in marrow fat in Vegfa
CKO mice is similar to changes associated with osteoporosis and age-related osteopenia (4
). Postmenopausal women with polymorphisms associated with high or low VEGF production have higher or lower lumbar spine bone mineral density, respectively (49
). Intriguingly, there also appears to be no significant association between levels of circulating VEGF and bone mineral density (49
). This is consistent with our in vitro data demonstrating that addition of recombinant VEGF to cultures of VEGF-deficient BMSCs, even at very high concentrations, has no effects on the differentiation properties of the mutant cells. This suggests that VEGF controls osteoblastogenesis and adipogenesis via mechanisms that are resistant to the effects of extracellular VEGF.
The concept of an intracrine signaling loop for VEGF has been previously suggested in the case of bone marrow hematopoietic stem cells (50
). VEGF appears to control survival of these stem cells via a mechanism that is resistant to inhibitors that fail to penetrate the plasma membrane, such as antibodies (50
). A similar phenomenon has been noticed in studies in which VEGF was specifically deleted in endothelial cells (51
). Furthermore, a recent report suggests that VEGF increases survival and chemoresistance in human colorectal cancer cells by an intracrine mechanism (52
). How intracrine functions of VEGF may be regulated and in which compartments VEGF may function intracellularly has not been known. However, several observations provide intriguing hints. VEGF has been reported to mediate survival of human breast cancer carcinoma cells via binding intracellularly to VEGFR1 (53
), and it has been found to accumulate in endothelial cell nuclei (54
). The immunocytochemical detection of sVEGFR1, VEGFR2, and VEGF in the nuclear region of BMSCs in the present study is consistent with these observations. A key question is whether the intracrine function in Osx-positive osteoblastic lineage cells requires direct binding of VEGF to its receptors. Our data do not allow a definitive answer to this question. However, since knockdown of either Flt1
in the osteoblast lineage led to reductions in the numbers of ALP-positive CFU-F colonies, interactions of intracellular VEGF with sVEGFR1 and VEGFR2 in differentiating osteoblasts may be part of the mechanism by which VEGF stimulates osteoblastic differentiation in BMSCs. In lysates of BMSCs, VEGFR2 was found to be phosphorylated on Y1175, and downstream targets, such as Akt and p38MAPK, were phosphorylated as well even when cell-associated VEGF protein levels were knocked down (Supplemental Figure 1). Therefore, while the receptors may be involved in the intracrine effects of VEGF on osteoblastic differentiation, it is unlikely that the effects are mediated by signaling downstream of activated VEGFR2. Interestingly, Domingues et al. (55
) recently reported that a VEGF/VEGFR2 complex can be transported into nuclei in endothelial cells, where it can interact with several nuclear proteins, including the transcription factor Sp1, and stimulate the expression of VEGFR2. If this mechanism also applies to BMSCs, one would expect the level of VEGFR2 to decrease with decreasing levels of VEGF in the cells, and this is consistent with lower levels of VEGFR2 in lysates from BMSCs treated with VEGF-specific shRNA.
The association between low levels of VEGF and low levels of RUNX2 protein and activity in BMSCs provides an explanation for the reduced ability of the cells to differentiate into osteoblasts when VEGF levels are knocked down. Elucidation of the mechanisms by which VEGF affects RUNX2 expression and activity will require further studies. However, since Sp1 has been demonstrated to be important for stimulation of Runx2
), the findings of Domingues et al. (55
) raise the possibility that VEGF/VEGFR2 complexes may have a role in Sp1-dependent induction of Runx2
transcription. Combined with evidence that RUNX2 can serve as a transcription factor to induce transcription of Vegfa
), this suggests the existence of a positive feedback loop between intracrine VEGF and RUNX2 expression.
The suppression of adipocyte differentiation by VEGF does not include VEGFR1- and VEGFR2-dependent signaling mechanisms. Results of CFU-A assays with VEGFR2-deficient BMSCs were not different from those of assays with control cells. Also, VEGFR1 deficiency in BMSCs inhibited adipogenesis. This is consistent with the known function of sVEGFR1 as a high-affinity decoy receptor for VEGF in many contexts; loss of VEGFR1 may increase the efficiency of VEGF as a suppressor of adipogenesis. Thus, other VEGF-binding receptors or receptor-independent VEGF functions may be involved in the mechanism by which VEGF suppresses adipocyte differentiation. Knockdown of lamin A in mesenchymal stem cells results in increased adipocyte differentiation (42
), raising the question of whether the effect of VEGF on adipocyte differentiation is mediated by lamin A. Our data indicate that this is not the case. Loss of one Lmna
allele in BMSCs caused cell-associated VEGF to be reduced to the levels in cells treated with VEGF-specific shRNA, suggesting that lamin A stimulates VEGF protein expression. In addition, the fact that levels of lamin A/C expression increased when WT or Lmna+/–
mesenchymal cells expressed VEGF-specific shRNA indicates that VEGF, in turn, suppresses lamin A expression. Such a reciprocal relationship between VEGF and lamin A may explain why loss of one Lmna
allele in cells results in lamin A levels that are greater than 50% of what they are in WT cells. The relationship between VEGF and lamin A also indicates that lamin A is upstream of VEGF with respect to both osteoblast and adipocyte differentiation. In fact, our data suggest that most, if not all, effects of lamin A on osteoblast and adipocyte differentiation may be mediated by the effects of lamin A on the level of VEGF in cells (Figure ).
Diagram illustrating a model for the reciprocal functional interactions between VEGF and lamin A and the proposed relationships among the levels of VEGF, RUNX2, and PPARγ2 based on the data.
Activation of Wnt/β-catenin signaling suppresses adipogenesis and promotes osteoblastogenesis of mesenchymal stem cells. This raises the question of whether Wnt signaling may be involved in the VEGF-dependent functions in the cells (13
). However, disruption of canonical Wnt signaling in mice causes bone loss due to reduced osteoblast differentiation, but adipogenesis is not affected (16
). Furthermore, transdifferentiation of mesenchymal stem cell–derived osteoblast progenitors into adipocytes is not associated with a change in expression of Wnt3a and β-catenin (58
In addition to the intracrine mechanism for VEGF function related to osteoblast/adipocyte differentiation as suggested by the data presented here, we also demonstrate that VEGF secreted by BMSCs has paracrine effects on osteoclast differentiation in assays using total bone marrow cell cultures. Furthermore, it is known that osteoblast-derived VEGF can affect bone formation in a paracrine manner via stimulation of angiogenesis (59
). It will be interesting to investigate whether and how osteoblast-derived intracrine VEGF affects the interaction between osteoblasts and endothelial cells in bone marrow, since it has been reported that direct contact between endothelial cells and osteoprogenitors in vitro supports osteoblast function (60
). To our knowledge, this is the first in vivo/in vitro study to investigate the roles of osteoblast-derived VEGF in osteoblastic and adipocytic differentiation. Based on the results of the study, we propose that understanding the mechanisms by which intracrine VEGF controls adipocyte and osteoblast fates may lead to identification of novel therapeutic targets to control bone loss in osteoporosis.