Notch Signaling Induces Osteogenic Differentiation and Mineralization of Vascular Smooth Muscle Cells
Role of Msx2 Gene Induction via Notch-RBP-Jk Signaling
Objective— Vascular calcification is closely correlated with cardiovascular morbidity and mortality. Here, we demonstrate the role of Notch signaling in osteogenic differentiation and mineralization of vascular smooth muscle cells (SMCs).
Methods and Results— The Msx2 gene, a key regulator of osteogenesis, was highly induced by coculture with Notch ligand-expressing cells or overexpression of Notch intracellular domains (NICDs) in human aortic SMCs (HASMCs). Furthermore, the Notch1 intracellular domain (N1-ICD) overexpression markedly upregulated alkaline phosphatase (ALP) activity and matrix mineralization of HASMCs. A knockdown experiment with a small interfering RNA confirmed that Msx2 mediated N1-ICD–induced osteogenic conversion of HASMCs. Interestingly, Msx2 induction by N1-ICD was independent of bone morphogenetic protein–2 (BMP-2), an osteogenic morphogen upstream of Msx2. The transcriptional activity of the Msx2 promoter was significantly enhanced by N1-ICD overexpression. The RBP-Jk binding element within the Msx2 promoter was critical to Notch-induced Msx2 gene expression. Correspondingly, N1-ICD overexpression did not induce the Msx2 expression in RBP-Jk–deficient fibroblasts. Immunohistochemistry of human carotid artery specimens revealed localization of Notch1, Jagged1 and Msx2 to fibrocalcific atherosclerotic plaques.
Conclusion— These results imply a new mechanism for osteogenic differentiation of vascular SMCs in which Notch/RBP-Jk signaling directly induces Msx2 gene expression and suggest its crucial role in mediating vascular calcification.
Vascular calcification is commonly seen with aging, end stage renal disease, diabetes, and atherosclerosis and is closely associated with cardiovascular morbidity and mortality.1,2 Once considered to be a passive and unregulated process, it is now known to be an active and tightly regulated phenomenon, in which a variety of osteogenic regulatory factors are involved.1–3 The presence of ossified bone within plaques and the expression of osteogenic cell markers including Msx2, Runx2, alkaline phosphatase (ALP), and osteopontin have been reported.4,5 In vitro studies have shown that vascular cells, including vascular smooth muscle cells (VSMCs), can undergo a phenotypic switch characterized by a loss of expression of SMC markers and a gain in osteoblastic phenotypes in response to factors such as elevated levels of inorganic phosphate (Pi) and bone morphogenetic proteins (BMPs).3,6 However, the precise molecular mechanisms of vascular calcification remain to be determined.
Among these osteogenic regulatory factors, Msx2 is considered to be a key regulator of vascular calcification.1,5 Msx2 was originally identified as a homeodomain transcription factor responsible for osteoblast differentiation and mineralization. Patients with a deletion or mutation of the Msx2 gene have skull bone defects, whereas a gain-of-function mutation gives rise to craniosynostosis, a condition characterized by premature fusion of the cranial sutures.7,8 In addition, increasing evidence indicates that Msx2 also modulates the formation of vascular calcification; BMP-2–Msx2 signaling promotes osteogenic mineralization of cultured myofibroblasts,9 and both BMP-2 and Msx2 are upregulated in aortic adventitial myofibroblasts in LDL receptor–null mice fed high-fat diets.10 Msx2-expressing adventitial myofibroblasts promote vascular calcification by producing Wnt agonists.11 Thus, Msx2 plays a critical role in vascular calcification.
The evolutionarily conserved Notch signaling pathway controls various cell fates by local cell–cell interactions.12 Notch ligands on the cell surface interact with Notch receptors in adjacent cells to cause cleavage of the Notch intracellular domain (NICD). NICD migrates into the nucleus and associates with RBP-Jk, which further activates transcription of target genes.13 Notch signaling has been implicated in the pathogenesis of vascular diseases, as well as in the embryonic development of the vasculature.13 In addition, recent studies have reported that Notch signaling is involved in osteoblastic differentiation of osteoblast precursor cells.14–17 Therefore, there is major interest in further elucidating the role of Notch signaling in the osteogenic conversion of VSMCs.
Here, we tested the hypothesis that Notch signaling plays a pivotal role in osteogenic conversion of VSMCs and the formation of vascular calcification. We show that canonical Notch-RBP-Jk signaling induces osteogenic conversion and mineralization of vascular SMCs through direct transcriptional activation of the Msx2 gene.
Materials and Methods
Alkaline Phosphatase Assay
ALP activity of various cells was measured using LabAssay ALP (Wako Pure Chemical Industries), according to the manufacturer’s protocol. ALP activity was normalized to total protein determined with a Bio-Rad protein assay solution (Bio-Rad Laboratories).
For a detailed Materials and Methods section, please see the supplemental materials (available online at http://atvb.ahajournals.org).
Both Stimulation With L-Jag1 and L-Dll4, and Overexpression of Notch Intracellular Domains Induced Msx2 Gene Expression in HASMCs
In view of recent evidence that Notch signaling is involved both in phenotypic modulation of VSMCs and osteo/chondrogenesis,14–20 we hypothesized that the Notch signaling pathway is involved in osteogenic conversion of HASMCs. Given that Msx2 plays a critical role in the osteogenic conversion of VSMCs,1,9,11 we first tested whether Msx2 expression was regulated by Notch signaling. In coculture experiments of HASMCs and L-GFP, L-Jag1, or L-Dll4, Msx2 gene expression was induced in HASMCs treated with L-Jag1 and L-Dll4, and such Msx2 induction was completely abolished by DAPT, a specific inhibitor of Notch signaling (data not shown). Consistently, adenovirus expressing ICDs of Notch1, 3, and 4, all of which are expressed in the vascular systems,13 induced Msx2 gene expression in HASMCs (Figure 1A and 1B). Similarly, Ad-N1-ICD induced Msx2 in C3H10T1/2 cells (supplemental Figure IIIA and IIIB). Taken together, these results suggest that activation of Notch signaling is sufficient for osteogenic conversion of HASMCs, and that Msx2 is the downstream target gene of Notch signaling.
Overexpression of N1-ICD Markedly Provoked ALP Activity and Matrix Mineralization of HASMCs
We next attempted to determine whether Notch signaling induces ALP activity, an early marker of osteogenic conversion in HASMCs. As determined by assays using BCIP/NBT, a substrate for ALP, Ad-N1-ICD markedly induced ALP activity of HASMCs and C3H10T1/2 cells (Figure 1C and 1D and supplemental Figure IIIC). Likewise, Ad-N3-ICD and AD-N4-ICD strongly induced the ALP activity (supplemental Figure II). Because ALP activity is functionally important in in vitro calcification,1,2 we next examined whether such high expression of ALP induced by N1-ICD overexpression can also cause matrix mineralization in HASMCs. Interestingly, dense calcium deposition in the extracellular matrix of HAMSCs was observed in Ad-N1-ICD–treated HASMCs (Figure 1E). Taken together, these findings suggest that Notch1 signaling, as assessed by ALP activity and Kossa staining, induces osteogenic differentiation and mineralization of HAMSCs.
Msx2, but not Runx2/Cbfa1, Mediated N1-ICD–Induced ALP Activity in HASMCs
Previous studies have identified key regulators responsible for osteogenic differentiation of VSMCs. The sodium-dependent phosphate transporters Pit-1 and Pit-2, and osteogenic transcription factor Runx2/Cbfa1, mediate phosphate-induced vascular calcification.3,21 Therefore, we tested whether these factors were also upregulated in HASMCs infected with Ad-N1-ICD. As shown in Figure 2A and 2B, expression of Runx2/Cbfa1 and its target gene Osteocalcin (OC) were not affected. Activity as well as expression of Pit1 and Pit2 (Figure 2A) appeared to be unchanged, given that expression of Runx2 was not altered. In contrast, Wnt3a and Wnt7a, 2 major Wnt agonists that mediate Msx2-induced vasculopathy,11 were upregulated (Figure 2C and 2D). These findings suggest that Msx2, but not Runx2/Cbfa1, is the downstream target gene of Notch1 signaling.
To directly test whether Msx2 or Runx2/Cbfa1 mediate Notch-induced osteogenic conversion of HASMCs, Msx2 or Runx2/Cbfa1 were specifically silenced using siRNA. As shown in Figure 2E, N1-ICD–induced ALP activity was found to be significantly inhibited in HASMCs transfected with siMsx2, but only modestly inhibited in HASMCs transfected with siRunx2 as compared with that in cells transfected with siGFP. These results indicate that Msx2, but not Runx2/Cbfa1, is required for Notch signaling-induced osteogenic differentiation of HASMCs.
N1-ICD Activated Msx2 Gene Expression Independent of BMP-2 Signaling
BMP-2 has been considered to be a crucial mediator of vascular calcification,2,4,6 and Msx2 is a direct target gene of BMP-2 that partially mediates the osteogenic effect of BMP-2.1 Therefore, we aimed to determine whether BMP-2 is involved in Notch1-induced activation of Msx2 gene expression. To this end, we first tested whether neutralization of BMP-2 affects inducible expression of the Msx2 gene by N1-ICD overexpression. Results showed that N1-ICD induction of Msx2 gene expression was not diminished, despite the presence of an anti-BMP2/4 neutralizing antibody in the culture medium (Figure 3A).
We next determined whether N1-ICD induces expression of BMP-2 in HASMCs or facilitates secretion of BMP-2 to the culture medium. Induction of ALP activity by N1-ICD was not accompanied by an induction of BMP-2 expression or secretion (Figure 3A through 3C). These findings suggest that BMP-2 is not involved in N1-ICD–induced Msx2 gene expression.
Notch Ligands and N1-ICD Induced Msx2 Promoter Activity
To test whether Notch signaling upregulates transcription of the Msx2 gene, we used luciferase reporter constructs, Msx2–3.2k-Luc and Msx2–5.1kΔ3.3k-Luc, which contain a murine Msx2 promoter region between −3212 and −1 and between −5082 and −3289, respectively (Figure 4A). Transient transfection assays of these constructs into RASMCs showed that luciferase activities of Msx2–5.1kΔ3.3k-Luc were increased by N1-ICD overexpression (Figure 4B). In contrast, p6OSE-Luc, which contains a Runx2 binding site within an Osteocalcin promoter, did not respond to N1-ICD overexpression (Figure 4C). These results suggest that Notch1 signaling activates Msx2 gene expression at the transcription level, and that Runx2-mediated signaling is not activated by Notch1.
RBP-Jk Was Required for N1-ICD–Induced Msx2 Expression
RBP-Jk is a major mediator of Notch signaling. In the nucleus, RBP-Jk associates with NICD and forms a complex that further activates transcription of target genes from its cognate DNA binding sequence, GTGGGAA (RBP-Jk binding site).13 Interestingly, we found a putative RBP-Jk binding site in the murine Msx2 gene promoter, 5′-TTCCCAC-3′, between −3794 and −3788 from the transcription start site. To confirm the involvement of this sequence in the regulation of Notch-induced Msx2 gene activation, we performed luciferase assays using a series of mutants (Figure 4D). As expected, Notch ligand-induced Msx2 promoter activity of the deletion mutants was dependent on the putative RBP-Jk binding site. Consistently, Msx2(mRBS)-Luc, which contains a base substitution within the RBP-Jk binding site in the context of Msx2–5.1kΔ3.3k-Luc, failed to be activated. These findings indicate that the putative RBP-Jk binding sequence mediates the Notch signaling-induced Msx2 activation.
Next, we tested whether RBP-Jk physically binds to the DNA sequence of the Msx2 promoter identified in Figure 4D. DNA affinity precipitation assays revealed that RBP-Jk specifically interacted with the putative RBP-Jk binding site of the Msx2 promoter (Figure 4E). Furthermore, mutation of the sequence abolished the binding of RBP-Jk, confirming that this specific sequence mediates transactivation of the Msx2 gene by Notch-RBP-Jk signaling.
To further determine the role of RBP-Jk in Notch-induced Msx2 activation, we used mouse embryonic fibroblasts, OT13 and OT11 cells, in which RBP-Jk is preserved and deficient, respectively. Although Ad-N1-ICD upregulated Msx2 expression in OT13 cells, as well as in HASMCs and C3H10T1/2 cells, it did not alter Msx2 expression in OT11 cells (Figures 1C and 4⇑F, supplemental Figure IIIA and IIIB). In addition, increased luciferase activity of Msx2–5.1kΔ3.3k-Luc induced by Notch ligand-expressing L cells was observed in OT13 cells, but not in OT11 cells (Figure 4G). These results demonstrate that RBP-Jk is necessary for induction of Notch signaling-mediated Msx2 gene activation.
Notch1, Jagged1, and Msx2 Are Expressed in Human Fibrocalcified Atherosclerotic Plaques
In normal human arteries and noncomplicated plaques obtained from human carotid arteries, expression of Notch1, Jagged1, and Msx2 was barely detected (data not shown). In contrast, as exemplified in Figure 5, strong signals for Notch1, Jagged1, and Msx2 were observed in a fibrocalcified lesion (type IV) (Figure 5C, 5D, and 5E). Notch1, Jagged1 and Msx2 were largely colocalized with each other but not with SM α-actin (Figure 5F), indicative of Notch1, Jagged1, and Msx2 expression in non SMCs. Of note, Notch1, Jagged1, and Msx2 expression were clearly detected in an area where apparent calcification was not observed, suggesting the role of Notch1-Msx2 pathways in the early stage of osteoblastic differentiation rather than the advanced stage of mineralization.
Notch Simultaneously Induces Osteoblast- and SMC-Marker Gene Expression in HASMCs
We have recently reported that N1-ICD overexpression induces SMC differentiation of embryonic fibroblast C3H10T1/2 cells and HASMCs.18 We tested whether N1-ICD simultaneously induces osteoblast- and SMC-marker gene expression in HASMCs. Expression of smooth muscle myosin heavy chain (SM-MHC) and SM α-actin genes were highly induced by N1-ICD in HASMCs under the same culture conditions as that used in osteogenic differentiation (Figure 6A). In addition, we tested whether N1-ICD induction of osteogenic gene expression is cell type–specific. In contrast to the induction of ALP activity in HASMCs and C3H10T1/2 cells, N1-ICD did not induce, or rather repressed, ALP expression in mouse osteoblast MC3T3-E1 cells (Figure 6B), indicating that the effects of N1-ICD on osteogenic gene expression are cell type–specific.
The results presented in this study imply that Notch signaling directly activates Msx2 gene expression, thus promoting osteogenic differentiation of vascular SMCs and contributing to vascular calcification. This conclusion is supported by several lines of observation. First, both stimulation with Notch ligand-expressing L cells and adenoviral overexpression of NICDs induced Msx2 gene expression in HASMCs. Particularly, N1-ICD overexpression induced osteogenic differentiation of HASMCs, as assessed by their ALP activity and matrix mineralization in the presence of a high Pi concentration. Second, osteogenic conversion was independent of either Runx2/Cbfa1 or BMP-2. Third, the RBP-Jk binding site within the Msx2 promoter mediated Notch-induced Msx2 gene expression. Fourth, Notch1, Jagged1, and Msx2 expression was observed in fibrocalcified human atherosclerotic plaques, and their expression were largely colocalized with each other.
Notch Signaling Directly Promotes Osteogenic Conversion of Vascular SMCs
There are conflicting reports about Notch signal-mediated osteoblast differentiation. The predominant view in the field of osteogenesis is that Notch signaling suppresses the osteoblastic differentiation of osteogenic progenitor cells,14,16,17 whereas several reports have shown that Notch promotes osteoblastic differentiation of multipotent mesenchymal cells.15,22 Our finding that activation of the Notch signaling cascade induced the osteoblastic phenotype of SMCs is consistent with the latter, and also conforms to the notion that Notch signaling, in addition to inhibiting cell fate, can serve to promote cell fate by upregulating “master control genes” such as eyeless (ey), vestigial (vg), and Distal-less (Dll), which induce the formation of eyes, wings, antennae, and legs in Drosophila.23
So, what are the possible explanations for the discrepant observations as to the role of Notch signaling in osteogenesis? As shown in Figure 6B, HASMCs and C3H10T1/2 showed increased ALP activity in response to N1-ICD, whereas N1-ICD decreased ALP activity in mouse osteoblastic MC3T3-E1 cells. These results suggest that activation of Notch signaling exerts different effects on cellular differentiation depending on the cell type, stage of cell development and experimental conditions, so called a “context dependency” characteristic of Notch signaling. More specifically, we can envisage that transcription factors responsible for cell type–specific and developmental stage–specific gene expression differentially interact with NICDs, and this should be examined in future studies.
RBP-Jk Is Essential for Notch-Induced Msx2 Expression
Previous experiments established the role of Msx2 as one of the key factors regulating vascular calcification. Towler and colleagues demonstrated that pericytes and adventitial myofibroblasts are diverted to the osteoblast lineage by Msx2-dependent transcriptional programming.1,9 BMP-2, a powerful bone morphogen expressed in vascular cells surrounding a highly calcifying center, is known to potently activate Msx2 gene expression through Smad signaling. Thus, Msx2 induction by BMP-2 appears to be a key mechanism of osteogenic differentiation of vascular SMCs. However, the possible role of Notch signaling in regulating Msx2 gene expression has not been documented. Here, we provide compelling evidence that Msx2 is a direct target gene for Notch/RBK-Jk signaling. We identified the RBP-Jk binding site 5′-TTCCCACA-3′ at −3794 from the transcriptional start site of the murine Msx2 gene and mutation of this sequence or RBP-Jk deficiency caused a near-complete loss of responsiveness to NICD.
What Are the Upstream Signals Evoking Notch-Mediated Msx2 Expression in Vascular SMCs?
Because Notch signaling is primarily initiated by interaction between Notch ligands and Notch receptors,13 followed by activation of the target genes, it is likely that ligand binding to Notch receptors triggers induction of Msx2 gene expression. Given that macrophages abundantly express Dll4 and Jagged1 in response to proinflammatory stimuli such as lipopolysaccharide (LPS), interferon-gamma, and interleukin-1 beta,24,25 and that the early-stage of vascular calcification is accompanied by macrophage infiltration,26 we speculate that direct cell–cell contact between macrophages and SMCs initiates the activation of Notch receptors and leads to the induction of Msx2 gene expression. Tintut et al previously reported that oxidized LDL–treated monocyte/macrophages exhibited significant ALP activity and mineralization in CVCs in coculture experiments.27 Although they did not determine its underlying molecular mechanism, their observations support our hypothesis indicating the role of cell–cell communication between Notch ligand/receptor–expressing cells and subsequent Notch–Msx2 pathway activation in the osteogenic conversion of vascular SMCs. In fact, Notch1 and Msx2 immunostaining was largely associated with CD68-positive areas (supplemental Figure V). Furthermore, our preliminary experiments showed that angiotensin II (Ang II) induced Jagged1 expression in human monocytic THP-1 cells, and the coculture of HASMCs with Ang II–treated THP-1 cells induced significant ALP activity in HASMCs (data not shown).
Loss of SM α-actin Expression in Fibrocalcifying Atherosclerotic Plaques
We observed that Notch1, Msx2, and Jagged1 expression correlated with each other, but were stained negative for SM α-actin in atherosclerotic plaques (Figure 5 and supplemental Figure V). This finding is consistent with the notion that vascular calcification is accompanied by downregulation of SMC-marker genes,3,5 but does not lend support to our hypothesis that SMCs undergo osteogenic differentiation by Notch signaling in human calcifying lesions. However, the possibility that Notch1 and Msx2 expression is induced in SMCs cannot be excluded because of the following reasons.
It is noteworthy that Runx2 was strongly expressed in Notch1- and Msx2-positive cells (Figure 5G), although the Runx2 gene is not activated by either Nocth1 or Msx2 signaling. Our recent study demonstrated that Runx2 acts as a repressor for SMC marker gene expression by inhibiting SRF/myocardin-dependent transcription.28 Thus, we reason that loss of SM α-actin expression in Nocth1- and Msx2-expressing cells is attributable to the elevated levels of Runx2 expression. This hypothesis will be supported by a recent report by Speer and Giachelli; they showed that, using a genetically modified animal model of vascular calcification, osteochondrogenic precursor and chondrocyte-like cells in calcifying blood vessel were of SMC origin, and that they were negative for SMC marker genes but positive for Runx2.29
In line with this view, we can reconcile the discrepant results between in vitro and in vivo observations with regard to the SMC-marker gene expression during Notch-induced osteogenic differentiation. As shown in Figure 6A, N1-ICD simultaneously induced osteogenic-marker and SMC-marker gene expression in HASMCs, inconsistent with a previous study showing that gaining the osteogenic phenotype is invariably coupled with loss of the SMC phenotype in vascular calcification.30 Unlike our observation in vivo, in which Runx2, as well as Notch1 and Msx2, were strongly expressed in fibrocalcifying atheroma lesions (type V lesion), NICD-induced osteogenic differentiation did not accompany the induction of Runx2 expression. Given that Runx2 acts as a repressor for SMC marker gene expression in HASMCs,28 we propose that the discrepant in vitro and in vivo results with regard to SMC-marker gene expression in osteoblastic cells may be due to the expression levels of Runx2. This hypothesis is illustrated in Figure 6C.
An alternative hypothesis to explain the difference in SMC-marker gene expression between in vivo and in vitro is that Notch signaling per se is not sufficient for cell lineage determination, but rather contributes to amplify or consolidate phenotypic switching in collaboration with environmental cues including growth factors, inflammatory cytokines, phosphate concentration, and oxidative stress. Correspondingly, our recent experiments showed that osteogenic BMPs, such as BMP-2 and BMP-4, markedly amplified Notch-induced osteogenic conversion (data not shown).
Which Types of Vascular Calcification Are Attributable to the Notch1-Msx2 Pathway?
Vascular calcification is histoanatomically classified into 4 general types1: atherosclerotic/fibrocalcific, cardiac valve, medial artery calcification, and vascular calciphylaxis. As for atherosclerotic/fibrocalcific calcification, Runx2 is thought to play a pivotal role in its formation.1 However, our in vitro data and immunohisitochemistry indicates that the Notch1-Msx2 pathway is also involved in osteogenic differentiation of SMCs within atheroma. In addition, in view of the fact that atherosclerotic/fibrocalcific calcification is associated with abundant infiltration of inflammatory cells and that macrophages express Notch ligands in response to inflammatory stimuli, Notch-dependent mechanisms are deemed to be at least partly responsible for vascular calcification in atheroma. Of importance, the Msx2 gene activation was independent of BMP-2, a master regulator of vascular calcification targeting Msx2, as well as Runx2/Cbfa1 (Figure 3).1 Thus, the Notch-Msx2 pathway may be a novel mechanism for osteogenic differentiation and vascular calcification distinct from conventional BMP-2 signaling. Particularly, this pathway may work at the initiation of vascular calcification, given the observation that Notch/Msx2 expression was observed in the area where mineralization was not yet developed (Figure 5). This hypothesis is consistent with the fact that Msx2 functions at the very early phase during bone formation and diminishes in later stages.31 Together with our finding that Notch alone is not sufficient, and that cooperation with a Pi-induced mechanism is required for mineralization, coexpression of Msx2 with Runx2 supports our hypothesis that Notch-Msx2 signaling and Pi-Runx2 signaling work independently, but cooperatively, in the formation of vascular calcification.
In summary, we demonstrated that Notch signaling promotes osteogenic differentiation and mineralization of vascular SMCs by directly activating Msx2 gene transcription via RBP-Jk. We also showed the colocalization of Notch1 and Msx2 within human atherosclerotic/fibrocalcific plaques. These findings provide novel insight into the role of the Notch-RBP-Jk–Msx2 signaling pathway in vascular calcification.
Sources of Funding
This work was supported in part by Grants-in-Aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) (to M.K.), Memorial Research Grant from The Japan Research Promotion Society for Cardiovascular Disease (to T.T.), and Initiatives for Attractive Education in Graduate School from MEXT (to T.S.).
Received August 12, 2008; revision accepted April 23, 2009.
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