Msx2 Promotes Osteogenesis and Suppresses Adipogenic Differentiation of Multipotent Mesenchymal Progenitors*

In the aorta, diabetes activates an osteogenic program that includes expression of bone morphogenetic protein- 2 (BMP2) and the osteoblast homeoprotein Msx2. To evaluate BMP2-Msx2 signaling in vascular calcification, we studied primary aortic myofibroblasts. These cells express vascular smooth muscle cell (VSMC) markers, respond to BMP2 by upregulating Msx2 , and undergo osteogenic differentiation with BMP2 treatment or tranduction with a virus encoding Msx2. The osteoblast factor osterix ( Osx ) is upregulated 10- fold by Msx2 but Runx2 mRNA is unchanged; the early osteoblast marker alkaline phosphatase increases 50-fold with mineralized nodule formation enhanced 30-fold. Adipocyte markers are concomitantly suppressed. To better understand Msx2 actions on osteogenesis vs. adipogenesis, mechanistic studies were extended to mesenchymal cells. α γ vs. of


Introduction
Mineral deposition in the skeleton is regulated by morphogenetic, metabolic, mechanical, inflammatory, and endocrine factors. With aging, abnormalities in orthotopic (e.g., bone formation) and heterotopic arterial vascular calcification are observed with very high prevalence (1) --the latter enhanced by hyperglycemia, hyperlipidemia, and chronic renal insufficiency (1), (2). At least three variants of vascular calcification have been described: (a) Calcification of necrotic, intimal atherosclerotic plaques; (b) medial artery calcification; and (c) calcific sclerosis of the aortic valve.
Vascular calcification is a highly significant complication of diabetes, and has emerged as a powerful predictor of cardiovascular morbidity and mortality (2). The molecular mechanisms that perturb normal vascular calcium metabolism are only beginning to be understood (1,3,4). Demer was the first to show that vascular calcification may progress via molecular processes similar to osteogenesis (5). This group showed that the powerful bone morphogen, bone morphogenetic protein 2 (BMP2) is expressed in calcified atherosclerotic plaques of humans (5). Bostrom et al further demonstrated that aortic calcification in response to matrix Gla protein (MGP) deficiency was most likely via BMP2 signaling; MGP can abrogate alkaline phosphatase (ALP) induction by inhibiting BMP2 association with the BMP receptor (6). Thus, these studies point to a role for BMP2 in vascular calcification.
The SFG retrovirus has been shown to have no effect on osteoblast differentiation (15,16). Pseudotyped retroviral particles were prepared as previously detailed (14,15).
First passage transduced cells were used for all the assays.
Osteogenic gene expression and mineralization assays --RNA extraction and RT-PCR was performed as detailed (7,17). The primers for Msx2, OPN, and GAPD were reported previously (7,10). Adipogenesis assays -Confluent cultures of transduced C3H10T1/2 cells were subjected to adipogenic medium containing 0.1 µM dexamethasone, 50 µM indomethacin, and 5 µg/ml insulin (DII medium) for 14 days. The progression of adipogenesis was monitored under light microscope. At the end of culture period, cells were stained for lipid droplets using Oil Red-O stain as described (12).
Statistics --Statistical analyses were performed using Student's unpaired t-test or oneway ANOVA as previously detailed (22). Each experiment was performed at least twice and the representative data were presented as mean ± SEM of independent replicates (n > 3).

BMP2-Msx2 signaling promotes osteogenic differentiation of vascular myofibroblasts. --
We previously demonstrated that high fat diabetogenic diets induce calcific vasculopathy and aortic Msx2 and osteopontin expression in LDLR-/-mice (7). To understand the mechanisms leading to aortic calcification, we analyzed effects of high fat diets on the expression of BMP2, a potent osteogenic agent (23) that activates Msx2 expression (9) and has been identified in human atherosclerotic plaques (5). As compared to mouse chow, high fat diabetogenic diets --either with (Fat + Chol) or without (Fat) cholesterol supplementation -concomitantly upregulated aortic BMP2 and Msx2 mRNA accumulation ( Fig.1 A; also ref. (7)). Msx2 expression in aorta is primarily localized to a sub-population of adventitial and valvular fibrosal cells that also expressed VSMC αactin (7). To confirm that BMP2-Msx2 signaling can facilitate aortic calcification, we studied effects of BMP2 and Msx2 on the commitment of aortic myofibroblasts to osteogenic differentiation. Primary aortic myofibroblasts (VSMC α-actin + ) were prepared as previously detailed (10). As shown in Figure 1B, BMP2 treatment of myofibroblasts for 8 days upregulated ALP activity, an early mineralization marker indicating commitment to osteogenic differentiation (16,18 Msx2 expression stimulates osteogenesis of myofibroblasts --Osx is a BMP2-induced transcription factor that directs osteoblast-specific differentiation, upregulates ALP expression, and is necessary for mineralization (24). To show that Msx2 enhances osteogenesis in myofibroblasts, we evaluated effects of Msx2 on Osx expression and mineralized nodule formation. As shown in Figure 3A, Msx2 upregulated Osx 10-fold; by contrast, expression of Runx2 --a transcription factor that demarcates the bipotential osteoblast and chrondrocyte progenitor (24) --was unchanged (Fig. 3B). Mineralized nodule formation was also increased 30 -fold by Msx2 ( Fig. 3C and 3D). VSMC phenotypic markers SM22α and VSMC α-actin were not suppressed (in fact increased ca. 3-to 4-fold; Fig. 3E). By contrast, the adipogenic marker, PPARγ was suppressed >90% by Msx2 (Fig. 3F). Thus, Msx2 enhances osteogenesis of aortic myofibroblasts as evidenced by Osx expression, ALP activation, and enhanced mineralized nodule formation. Adipogenic markers such as PPARγ are concomitantly suppressed (vide infra).

Msx2 enhances osteogenic differentiation of C3H10T1/2 cells via mechanisms dependent
upon intrinsic Msx2 DNA binding activity --C3H10T1/2 is a well-characterized multipotential cell line for studying mesenchymal cell differentiation (12,20). To better shown in Figure 9A, protein -protein interactions between Msx2 and C/EBPα were readily identified in GST pulldown assays; radiaolabeled C/EBPα binds to GST-Msx2 (lanes 4 and 5) but not to GST resin (lanes 2 and 3). The interaction is specific, since C/EBPα does not directly bind to GST-Runx2 (lanes 6 and 7). The functional consequence of the interaction was next addressed. As shown in Figure 9B,  (Fig. 9C). We wished to assess the specificity of this functionally antagonistic interaction between Msx2 and other C/EBP family members. Therefore, a series of C/EBP expression plasmids were tested for the capacity to reverse Msx2-dependent suppression of the PPARγ promoter. The pcDNA3-C/EBP plasmids were used at 10-fold higher concentrations than the pcDNA-Msx2 expression plasmid to accentuate functional differences between the C/EBP family members in these experiments. As shown in Figure 9D, Msx2 co-expression suppresses the activity of the PPARγ promoter, which is overcome by C/EBPα, and to a lesser extent, by C/EBPβ / LAP (Fig. 9D). This effect is specific, since other C/EBP members such as LIP and C/EBPδ could not overcome Msx2-dependent suppression (Fig. 9D).
Thus, Msx2 inhibits expression of the adipogenic differentiation program in part via inhibitory protein -protein interactions with C/EBPα.

Discussion
Vascular calcification has emerged as an important harbinger of cardiovascular morbidity and mortality, particularly so in the setting of diabetes (2,28) -an increasingly prevalent disease achieving epidemic proportions (29). Decreased vascular compliance and increased arterial matrix remodeling in response to calcium deposition may contribute to disease pathophysiology and amputation risk (2,30). As compared to our understanding of orthotopic mineral metabolism, regulation of heterotopic vascular calcification is poorly understood (3). The hierarchy of pathophysiologically relevant signals that control vascular inflammation, smooth muscle proliferation, matrix remodeling, osteogenic differentiation, and mineral deposition remains to be determined via detailed study of appropriate in vivo and ex vivo model systems.
We previously reported that high fat diabetogenic diets promote hypercholesterolemia and vascular calcification in LDLR -/-mice, with concomitant upregulation of aortic Msx2 gene expression (7). By in situ hybridization, Msx2 is localized to adventitial cells and aortic valve fibrosal cells in a pattern overlapping that of VSMC α-actin (7). We speculated that this cell population might be a microvascular smooth muscle cell called a pericyte (31).  (39). Of note, the weaker transactivator C/EBPβ appears to play a more important role early on during adipogenic commitment (19). Given the stage-specific roles for C/EBP family members during adipogenesis (19)