Heat Shock Protein 70 Enhances Vascular Bone Morphogenetic Protein-4 Signaling by Binding Matrix Gla Protein
Rationale: Matrix Gla protein (MGP) is a calcification inhibitor, which binds and inhibits bone morphogenetic protein (BMP)-2 and -4.
Objective: The objective was to determine whether MGP also binds other proteins, which could interfere with its function.
Methods and Results: We transfected bovine aortic endothelial cells with N-terminally FLAG-tagged MGP and used immunoprecipitation and liquid chromatographic–tandem mass spectrometric analysis to identify MGP-binding proteins. Heat shock protein (HSP)70, a stress-induced protein expressed in atherosclerotic lesions and soluble in serum, was identified as a novel MGP-binding protein. The interaction between MGP and HSP70 was confirmed by coimmunoprecipitation and chemical crosslinking, and blocked the interaction between MGP and BMP-4. In endothelial cells, HSP70 enhanced BMP-4–induced proliferation and tube formation, and in calcifying vascular cells, HSP70 enhanced BMP-induced calcium deposition. In addition, HSP70 mediated the procalcific effect of interleukin-6 on calcifying vascular cells. In apolipoprotein E–null mice, a model for atherosclerosis, levels of BMP-4, HSP70, MGP, and interleukin-6 were elevated in the aortic wall. Levels of BMP-4, HSP70, and interleukin-6 were also elevated in serum, and anti-HSP70 antibodies diminished its procalcific effect on calcifying vascular cells.
Conclusion: HSP70 binds MGP and enhances BMP activity, thereby functioning as a potential link between cellular stress, inflammation, and BMP signaling.
Matrix Gla protein (MGP) is best known as an inhibitor of arterial calcification.1 It is a secreted protein expressed in the vascular endothelium and media2–4 that binds and inhibits the activity of bone morphogenetic protein (BMP)-2 and -4.1,5 MGP prevents BMP-2–induced mineralization in multipotent C3H10T1/2 cells and calcifying vascular cells (CVCs) in vitro,1,6 but it also prevents BMP-4 activity in endothelial cells in vitro, and pulmonary vascular development in vivo.5,7 Recently, we showed that the ability of MGP to bind BMP-4 depends on the presence of the proline-64 (Pro64) residue and γ-carboxylated glutamate residues (so-called Gla residues) in the MGP protein.8 It is not known, however, whether MGP is able to bind additional proteins.
The heat shock proteins (HSPs) were initially identified as intracellular proteins, which facilitate protein refolding, chaperone proteins and increase during stress to provide cellular protection.9,10 However, it is now known that HSPs, in particular HSP70 (also referred to as HSP72 and HSPA1A), are released to the extracellular milieu and circulation in response to stressful stimuli, where they exhibit potent immunomodulatory effects.11–13 The HSPs have also been implicated in atherosclerosis.10,14 In humans, high levels of HSP70 appear to have atheroprotective effects,14 and antibodies to HSP70 have been reported increased in patients with vascular disease.10 However, in apolipoprotein E–null (Apoe−/−) mice, in which tissues under stress are more easily defined because the stress-inducible form of HSP70 is not constitutively expressed, HSP70 levels correlate with lesion severity.15 Overexpression of HSP70 are found in several cell types in the lesions including endothelial cells and smooth muscle cells,10,15 but the precise role performed by HSP70 remains unclear.
In this study, we identified HSP70 as a MGP-binding protein and hypothesized that this interaction would affect BMP signaling because of the role of MGP as a BMP inhibitor. Our results showed that HSP70 enhanced the effect of BMP-4 on endothelial and medial vascular cells, and mediated a procalcific effect of interleukin (IL)-6, an inflammatory cytokine,16 in vitro. Together with BMP-4 and MGP, HSP70 was detected at increased levels in the aortas and sera of fat-fed Apoe−/− mice, a model for atherosclerosis, and neutralization of HSP70 in serum decreased its procalcific effect. The results suggest that HSP70 may function as a potential link between cellular stress, inflammation, and BMP signaling.
Cell Culture and Transfection Assays
Bovine aortic endothelial cells (BAECs) were cultured and transfected as previously described.5 Luciferase assays were performed as described previously and normalized to Renilla luciferase.5 Human aortic endothelial cells (HAECs) were cultured and transiently transfected with small interfering (si)RNA as described previously.5 Conditioned media from HEK293 cells transfected with N-FLG-MGP construct were prepared as previously described.8 Bovine CVCs were isolated and cultured as previously described.6 Transfection of CVCs with siRNA were performed as previously described17 using the Amaxa Nucleofector and the human AoSMC Nucleofector kit. The siRNAs for human and bovine HSP70 were obtained from Applied Biosystems (Foster City, Calif) as part of TaqMan Gene Expression Assays. Recombinant human BMP-2, BMP-4, and IL-6 (R&D Systems, Minneapolis, Minn), recombinant HSP70 and anti-HSP70 antibodies (Stressgen, Ann Arbor, Mich), and conditioned media with N-FLAG-MGP were added at the time of transfection or plating.
Wild-type and Apoe−/− mice on C57BL/6J background (The Jackson Laboratory, Bar Harbor, Me) were fed a standard chow or a high-fat/high-cholesterol diet (Western diet) (Research Diets, D12079B) for 16 weeks, starting at 8 to 10 weeks of age. Aortas and sera were collected for analysis. All procedures were conducted in accordance with the animal care guidelines set by the University of California, Los Angeles.
See the Online Data Supplement, available at http://circres.ahajournals.org, for the BMP-responsive element reporter gene (BRE-Luc) and the expression constructs for FLAG-tagged and mutated human MGP proteins.
Liquid Chromatography–Tandem Mass Spectrometry
BAECs were transfected with an expression construct for N-FLAG-MGP or empty control vector. Cells lysates were prepared 24 hours after transfection, and immunoprecipitated with anti-FLAG antibodies. The immunoprecipitated complexes were analyzed by SDS-PAGE gels and stained with SYPRO Ruby. In-gel digestion of proteins was performed as previously described (see the Online Data Supplement).18,19 Protein identification was accomplished by liquid chromatography–electrospray ionization–tandem mass spectrometry using an LTQ linear ion trap mass spectrometer (Thermo Electron) with a dedicated Surveyor pump system equipped with a reversed phase HPLC column (75 μm×10 cm, BioBasic C18, 5 μm particle size, New Objective, Woburn, Mass) as described in detail in the expanded Methods section in the Online Data Supplement. Spectra were acquired in data-dependent mode and searched against the human database using SEQUEST. All proteins were identified on the basis of 2 or more peptides. Criteria for positive identification include: XCorr values of >3.0 (+1), >4 (+2), and >5 (+3); >2 sequenced peptides; and DeltaCn >0.1. All spectra used for identification were manually inspected to ensure that the most abundant peaks were assigned.
Immunoprecipitation, Immunoblotting, and Crosslinking
Coimmunoprecipitation, immunoblotting, and crosslinking were performed using previously described methods (see the Online Data Supplement for details).5,8
Immunohistochemistry and ELISA
Immunohistochemistry were performed as described previously7,17 using antibodies to BMP-4 (R&D Systems), HSP70 (Stressgen), MGP,5 pSMAD1/5/8 (Cell Signaling Technology, Danvers, Mass), pSMAD2/3 (Cell Signaling), and total SMAD (Santa Cruz Biotechnology, Santa Cruz, Calif). ELISA for BMP-4 was from R&D Systems.
Proliferation and Tube-Formation Assays
Cell proliferation was determined by 3H-thymidine incorporation, and tube formation was determined by Matrigel assays (see the Online Data Supplement for details).
Alkaline Phosphatase Assay and Quantification of Calcium Deposition
Alkaline phosphatase (ALP) activity and calcium deposition were quantified as previously described.6,8
Data were analyzed for statistical significance by 2-way ANOVA with post hoc Tukey’s analysis using the GraphPad Instat 3.0 software (GraphPad Software, San Diego, Calif). Probability values of <0.05 were considered significant. All experiments were repeated a minimum of 3 times.
Identification of HSP70 As a MGP-Binding Protein
To identify MGP-binding proteins, we transfected BAECs with an expression construct for N-FLAG-MGP or an empty control vector, and collected cell lysates and media 24 hours after transfection. The FLAG-tagged MGP was used because it was easier to perform immunoprecipitation and immunoblotting with anti-FLAG antibodies; previous studies showed no functional effect of the FLAG-tag.5,8 We first studied cell lysates because we previously found MGP closely associated with the cells,20 suggesting that extracellular MGP functions close to the cell surface. The immunoprecipitated complexes were analyzed by SDS-PAGE gels and stained with SYPRO Ruby. Six bands, detected at 11, 15, 40, 70, and 200 kDa, were unique for samples from cells expressing N-FLAG-MGP (Figure 1A). These bands were extracted and tryptic peptides were analyzed by liquid chromatography–tandem mass spectrometry. The data-dependent spectra were compared to bovine, mouse and human databases. The proteins that were identified in all three databases as highly identical proteins are listed in Figure 1B. Of the proteins identified in this initial screen, we further pursued HSP70 because it has been strongly associated with vascular disease,10,14 and shown to be active extracellularly.11–13
In the next step, we tested whether the interaction was truly between extracellular MGP and HSP70 and whether it could be detected in the medium from nontransfected cells. Ten ml of BAEC-conditioned medium was immunoprecipitated using anti-MGP antibodies or control IgG, and analyzed by SDS-PAGE gels. SYPRO Ruby protein stain revealed two bands, which corresponded in size to HSP70 (70 kDa) and MGP (10.5 kDa) (Figure 1C), and reacted with anti-HSP70 and anti-MGP antibodies, respectively (Figure 1C). This supports an interaction between extracellular HSP70 and MGP.
To further confirm the interaction between HSP70 and MGP, lysates and media from nontransfected cells and cells transfected with N-FLAG-MGP were coimmunoprecipitated with anti-MPG and anti-FLAG antibodies, respectively, and analyzed by immunoblotting using anti-HSP70 antibodies. Conversely, anti-HSP70 immunoprecipitates were analyzed by immunoblotting with anti-MGP or anti-FLAG antibodies. In all cases, MGP and HSP70 coimmunoprecipitated (Figure 1D).
Finally, we performed crosslinking experiments using medium from BAECs transfected with N-FLAG-MGP. HSP70 (60 to 100 ng) was added to 80 μL of serum-free medium containing N-FLAG-MGP (≈50 ng/mL),5 and crosslinked with disuccinimidyl suberate. The complexes were analyzed by immunoblotting using anti-FLAG or anti-HSP70 antibodies. Crosslinking of HSP70 and N-FLAG-MGP resulted in the formation of a complex that migrated at ≈100 kDa on immunoblotting using both anti-FLAG (Figure 1E) and anti-HSP70 antibodies (Figure 1F). N-FLAG-MGP and HSP70 migrated at ≈10 and 70 kDa, respectively. Although no additional bands were detected between HSP70 and the HSP70-N-FLAG-MGP complex, the change in migration suggested that more than one MGP bound to HSP70. Our previous crosslinking studies suggested that a dimer of MGP bound to a dimer of BMP-4 (Figure 2B, lane 4, middle).8 Thus, the change in migration of HSP70 after crosslinking could be accounted for by one or two dimers of MGP. Taken together, the results strongly supported an interaction between HSP70 and MGP.
Binding of HSP70 to MGP Blocked Its Binding to BMP-4
We and others have previously shown that MGP binds and inhibits BMP-2 and -4.1,5,8 To determine whether HSP70 interfered with the binding between BMP-4 and MGP, we performed coimmunoprecipitation experiments. We added BMP-4, HSP70 or both to conditioned media containing N-FLAG-MGP (Figure 2A, lanes 1 to 4), and immunoprecipitated with anti-FLAG, anti-HSP70 or anti-BMP-4 antibodies. The precipitated complexes were then analyzed by immunoblotting; if one of the antibodies was used for immunoprecipitation, the other 2 antibodies were used for immunoblotting. The results revealed that coimmunoprecipitation of N-FLAG-MGP and BMP-4 occurred when HSP70 was absent (Figure 2A, lane 5). Addition of HSP70 abolished the coimmunoprecipitation of MGP and BMP-4 (Figure 2A, lane 7). However, coimmunoprecipitation of N-FLAG-MGP and HSP70 occurred regardless of whether or not BMP-4 was present (Figure 2A, lanes 7 and 8), suggesting that HSP-70 blocked the binding between BMP-4 and MGP. No interaction was seen between BMP-4 and HSP70 (Figure 2A, lane 6).
To validate our results, we performed crosslinking experiments. BMP-4, HSP70, or both were added to serum-free conditioned media containing N-FLAG-MGP (Figure 2B, lanes 1 to 3) and crosslinked. Immunoblotting with anti-FLAG antibodies revealed that incubation of BMP-4 and MGP led to the formation of the previously reported 62 kDa complex (Figure 2B, lane 4, top),8 corresponding to a BMP-4 dimer bound to a MGP dimer. Incubation of HSP70 and MGP led to the formation of a 100 kDa complex as before (Figure 2B, lane 7, top). Incubation of all 3 proteins led to the formation of 100-kDa complex only (Figure 2B, lane 6, top), suggesting that MGP preferentially bound to HSP70. Immunoblotting with anti–BMP-4 antibodies only detected complexes of BMP-4 and MGP (Figure 2B, lane 4, middle). Addition of HSP70 abolished the formation of BMP4-MGP complexes (Figure 2B, lane 6, middle). No crosslinked complexes of BMP-4 and HSP70 were detected (Figure 2B, lane 5, middle). Immunoblotting with anti-HSP70 antibodies also detected the 100-kDa complex of crosslinked HSP70 and MGP regardless of whether or not BMP-4 was present (Figure 2B, lanes 6 to 7, bottom). Taken together, the results supported that the binding between BMP-4 and MGP was abolished by HSP70.
Pro64 and Gla Residues Were Required for the Binding Between HSP70 and MGP
We previously showed that an intact Pro64 and at least one Gla residue on each side in the multi-Gla domain of MGP was critical for the binding between BMP-4 and MGP.8 To determine whether these residues were also essential for the binding between MGP and HSP70, we used two mutant versions of N-FLAG-MGP that are unable to bind BMP-4. N-FLAG-MGP-P64G contained a glycine instead of proline in position 64, and N-FLAG-MGP-4GlaG contained 4 glycine residues instead of 4 glutamate/Gla residues (Figure 3A, top).8 HSP70 was added to control medium or conditioned media containing N-FLAG-MGP, -MGP-P64G, or -MGP-4GlaG and coimmunoprecipitated with anti-FLAG or anti-HSP70 antibodies. Anti-FLAG immunoprecipitates were analyzed by immunoblotting using anti-HSP70 antibodies and vice versa. No coimmunoprecipitation was detected for N-FLAG-MGP-P64G or -MGP-4GlaG (Figure 3A), suggesting that Pro64 and Gla residues were essential for the interaction between HSP70 and MGP.
We also performed crosslinking experiments to validate the results. HSP70 was added to conditioned media containing N-FLAG-MGP, -MGP-P64G, or -MGP-4GlaG and crosslinked with disuccinimidyl suberate. The crosslinked complexes were analyzed by immunoblotting using anti-FLAG or anti-HSP70 antibodies. No crosslinking was detected between the mutant N-FLAG-MGP proteins and HSP70 (Figure 3B), which supported that the Pro64 and Gla residues were required for binding between HSP70 and MGP. Thus, HSP70 likely binds to the same MGP domain as BMP-4 thereby blocking the binding between MGP and BMP-4.
Depletion of HSP70 Did Not Affect Expression and Secretion of MGP
Although HSP70 is known to act as a chaperone protein,21 we were unable to detect an effect on expression and secretion of MGP from a depletion of HSP70 (Online Figure I).
Extracellular HSP70 Abolished the Inhibitory Effect of MGP on BMP-4 Activity
We have previously shown that MGP inhibits BMP activity in vascular cells.5,6 To determine whether HSP70 interfered with BMP signaling, we cotransfected BAECs with a BMP-responsive luciferase reporter gene (BRE-Luc) and increasing amounts of N-FLAG-MGP construct (0 to 200 ng plasmid/well). The cells were treated with BMP-4 alone (40 ng/mL) or in combination with HSP70 (1 or 10 ng /mL) for 24 hours. Luciferase assays showed that the BMP-4 activation of BRE-Luc was inhibited by MGP but that 1 ng/mL HSP70 partially recovered and 10 ng/mL HSP70 fully recovered BMP-4 activity (Figure 4A). HSP70 had no significant effect on endogenous MGP-expression (data not shown). We repeated the experiments using the N-FLAG-MGP-P64G and -MGP-4GlaG vectors, but the BMP-4 activity was unaffected by both the increased MGP levels and the added HSP70 (Figure 4A). We also cotransfected BAECs with BRE-Luc and the N-FLAG-MGP construct or empty vector (200 ng/well) and treated with BMP-4 (40 ng/mL) in combination with increasing amounts of HSP70 (0 to 10 ng/mL) for 24 hours. This revealed that HSP70 mildly enhanced BMP signaling in absence or presence of exogenous BMP-4 (Figure 4B) but significantly recovered BMP-4 activity in presence of MGP (Figure 4B). Again, N-FLAG-MGP-P64G and -MGP-4GlaG had no effect on BMP-4 activity and were unaffected by HSP70 (Figure 4B). The levels of the FLAG-tagged MGP proteins were similar as determined by immunoblotting (compare Figure 4C, bottom). Together, the results support that both the inhibition by MGP and stimulation by HSP70 of BMP-4 activity depend on a MGP protein that is able to bind BMP-4 and HSP70.
Extracellular HSP70 Enhanced BMP-4–Induced Activation of Endothelial Cells
BMP-4 is known to stimulate EC proliferation and vascular formation,22 and we therefore determined the effect of MGP and HSP70 on BMP-4–induced EC proliferation. We transfected BAECs with empty vector, N-FLAG-MGP, -MGP-P64G, or -MGP-4GlaG and treated with BMP-4 (40 ng/mL) in absence of presence of HSP70 (10 ng/mL). The expression of the respective MGP proteins was similar (Figure 4C). 3H-Thymidine incorporation revealed that BMP-4 increased proliferation, and that N-FLAG-MGP inhibited this effect (Figure 4C). However, HSP70 enhanced proliferation in absence and presence of exogenous BMP-4, and counteracted the effect of MGP (Figure 4C). N-FLAG-MGP-P64G and -MGP-4GlaG were indistinguishable from empty vector (Figure 4C).
To determine the effect of MGP and HSP70 on tube formation induced by BMP-4, we cultured BAECs on Matrigel in conditioned media from BAECs transfected with empty vector, N-FLAG-MGP, -MGP-P64G, or -MGP-4GlaG (MGP levels ≈40 ng/mL) and treated with BMP-4 (40 ng/mL) in absence or presence of HSP70 (10 ng/mL). The results showed that BMP-4 increased and MGP decreased tube formation (Figure 4D). Similar to proliferation, HSP70 enhanced tube formation and counteracted the effect of MGP (Figure 4D). Together, the results suggested that HSP70 enhanced BMP-4 activity in ECs. Again, the mutated MGP proteins had no effect on BMP-4 activity (Online Figure II).
Extracellular HSP70 Enhanced BMP-2 and BMP-4–Induced Cell Condensation and Osteogenic Differentiation in CVCs
MGP inhibits formation of cell condensation and mineralization induced by BMP-2 and -4 in CVCs, a well-established in vitro model for vascular calcification.1,6 To determine whether HSP70 blocked the activity of MGP in CVCs, we treated CVCs with BMP-4 (50 ng/mL) together with HSP70 (50 ng/mL) and/or conditioned medium containing N-FLAG-MGP (≈50 ng/mL, visualized by immunoblotting with anti-FLAG-antibodies, Figure 5B) for 2 or 8 days. After 2 days, BMP-4 induced formation of condensations (Figure 5A), and activity of ALP, an early osteogenic marker (Figure 5B). After 8 days, BMP-4 increased mineralization, a late osteogenic marker (Figure 5C). N-FLAG-MGP alone inhibited all three parameters (Figure 5A through 5C), whereas HSP70 alone had a mildly stimulating effect, likely attributable to enhancement of endogenously expressed BMP (Figure 5A through 5C).6 When BMP-4 was added together with MGP, the stimulating effect of BMP-4 was inhibited. However, addition of HSP70 further enhanced the BMP-4 effect on condensation, ALP activity and mineralization (Figure 5A through 5C), and if added together with N-FLAG-MGP, it neutralized the inhibitory effect of MGP (Figure 5A through 5C).
Because MGP is also known to inhibit BMP-2,6,20 we determined whether replacing BMP-4 with BMP-2 (300 ng/mL) would give similar results. The higher concentration of BMP-2 was attributable to lower bioactivity per ng compared to BMP-4. Indeed, condensation formation (not shown), ALP activity and mineralization results were similar to those obtained with BMP-4 (Figure 5D and 5E). We also replaced N-FLAG-MGP with -MGP-P64G or -MGP-4GlaG and examined condensation formation induced by BMP-4 (Online Figure III), as well as ALP activity and mineralization induced by BMP-2 and BMP-4 (Online Figure IV). The results showed that the mutated MGP proteins had no effect on BMP activity. Altogether, the results supported that HSP70 enhanced both BMP-2 and -4 signaling by diminishing the inhibitory effect of MGP.
IL-6–Induced Condensation and Osteogenic Differentiation in CVCs Is Mediated by HSP70
IL-6 is an inflammatory cytokine, which is known to induce mineralization in CVCs23 and expression of HSP70 in other cells.24 To determine whether IL-6 induced HSP70 in CVCs, the cells were treated with IL-6 (10 or 50 ng/mL) for 24 hours. The results showed that IL-6 increased media levels of HSP70 in media, as determined by immunoblotting (Figure 6A). We then examined whether IL-6 and HSP70 affected BMP signaling in CVCs and whether HSP70 mediated the IL-6 effect on condensation formation and osteogenic differentiation. The CVCs were treated with IL-6 (50 ng/mL) in the absence or presence of neutralizing anti-HSP70 antibodies (300 ng/mL) or control IgG. BMP signaling was determined after 12 hours by immunoblotting for phosphorylated (p)SMAD1/5/8 and compared to pSMAD2/3 (known to mediate transforming growth factor β signaling) and total SMAD proteins. Condensation formation and ALP activity were determined after 2 days and calcium accumulation after 8 days. The results showed that IL-6 promoted BMP signaling as well as the other parameters and that the anti-HSP70 antibodies abolished these effects (Figure 6B through 6E). It suggested that HSP70 mediated the enhanced BMP signaling and the procalcific effect of IL-6. To confirm the procalcific effect, we depleted the CVCs of HSP70 using HSP70 siRNA, which decreased the HSP70 level to less than 10% compared to scrambled control siRNA (Figure 6F). The cells were treated with IL-6 (50 ng/mL) starting 24 hours after transfection, and ALP activity and calcium accumulation were determined after 2 and 8 days, respectively. The depletion of HSP70 abolished the stimulating effect of IL-6 on both ALP activity and mineralization (Figure 6G and 6H), suggesting that HSP70 may mediate procalcific effects of IL-6 in the artery wall.
Increased Levels of BMP-4, HSP70, and MGP Increased in Apoe−/− Mice
High levels of HSP70 were previously described in atherosclerotic lesions in Apoe−/− mice.15 To compare vascular expression of HSP70 to that of BMP-4, MGP, and IL-6, aortas and sera were collected from wild type and Apoe−/− mice fed regular chow or a high fat diet for 16 weeks. Aortic expression of BMP-4, HSP70, MGP, and IL-6 was elevated in both chow- and fat-fed Apoe−/− mice, as determined by real-time PCR and immunoblotting (Figure 7A). (We were unable to perform immunoblotting for MGP in aortic extract.) Serum levels of BMP-4, HSP70 and IL-6 were also elevated, as determined by ELISA and immunoblotting (Figure 7B). Analysis of the Apoe−/− aortas by immunohistochemistry revealed that the expression of all 3 proteins was strongest in proximity of the endothelium in chow-fed Apoe−/− mice (Figure 7C, top images), whereas expression was found both close to the endothelium and in atherosclerotic lesions in fat-fed Apoe−/− mice (Figure 7C, bottom images). We also found that staining for pSMAD1/5/8 was increased in areas positive for HSP70, supporting an increase in BMP signaling (Online Figure V).
We then determined the effect of sera from wild type and Apoe−/− mice on chow or high-fat diet on BMP signaling and calcification in CVCs. BMP signaling was examined after 6 hours of incubation with 20% mouse serum using pSMAD1/5/8 immunoblotting. ALP activity and calcium deposition were determined after 2 and 8 days incubation, respectively, with 20% serum alone or in combination with neutralizing antibodies to HSP70 antibodies (300 ng/mL) or the BMP inhibitor Noggin (300 ng/mL). BMP signaling, ALP activity, and calcium deposition were all stimulated in CVCs incubated with serum from Apoe−/− mice, especially fat-fed mice (Figure 8A through 8C). However, anti-HSP70 antibodies and Noggin diminished the procalcific effect of the mouse sera (Figure 8B and 8C), suggesting that HSP70 and BMP both contribute to vascular calcification.
In this study, we provide evidence that HSP70 directly interacts with MGP, a BMP inhibitor. The interaction is dependent on the presence of proline-64 and Gla residues in the MGP-protein and blocks the interaction between BMP-4 and MGP, thereby enhancing BMP-4 signaling. The enhancement of BMP signaling explains the effect of HSP70 on proliferation and tube formation in ECs and on mineralization in CVCs in vitro.
We initially used cell lysates to identify the interaction between MGP and HSP70 because we had observed that MGP accumulates close to cells20 and speculated that it functioned close to the cell membrane. We confirmed that the interaction occurred in cell lysate and media from nontransfected cells using antibodies to native MGP, which supports that the interaction is physiological rather than secondary to overexpression of FLAG-tagged MGP. It also supports that the HSP70-MGP interaction occurs extracellularly and may be more important than the intracellular interaction based on the lack of effect on MGP secretion of HSP70 depletion. Interestingly, HSP70 is known to bind cell surface receptors such as LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) in endothelial cells,25 which may be affected by the HSP70-MGP interaction.
Even though HSP70 has been identified as a chaperone protein able to bind several proteins, and may have a higher likelihood for nonspecific interactions, it is unlikely that the interaction between HSP70 and MPG is nonspecific. Mutation of 1 amino acid (Pro64) or the Gla residues, the same residues required for interaction between MGP and BMP-4,8 abolished the HSP70-MGP interaction. Furthermore, the HSP70-MGP interaction blocked the BMP-4–MGP interaction. If HSP70 were generally “sticky,” HSP70 would have been more likely to bind the BMP-4–MGP protein complex, and we would have seen the presence of all 3 proteins in 1 complex. In addition, the mutated MGP proteins are unaffected by HSP70 in cell assays, further supporting specificity in the interaction.
We used 2 vascular cell models to study the cellular effects of altered BMP activity, aortic endothelial cells and CVCs. In both cases, the results showed that the BMP activity was enhanced by HSP70. In the endothelial cells, HSP70 enhanced BMP-4–induced cell proliferation and tube formation, whereas in CVCs, it enhanced ALP activity and calcium accumulation, which strongly supports that HSP70 is a regulator of BMP activity. Expression of BMP-2 and -4 is induced in endothelial cells by oscillatory shear stress and inflammatory mediators and appears to have a proatherogenic and proinflammatory effect in vitro.4,26,27 However, BMP-2 and -4 also stimulate the expression of MGP,5,17 and the same stimuli that induce BMPs also induce BMP inhibitors,4 making it difficult to predict whether BMP or BMP inhibitors ultimately will dominate. Thus, depending on the context, HSP70 may initially enhance BMP signaling, and then increase BMP-inhibition as a secondary effect.
HSP70 is known to be induced by inflammatory mediators28 and is expressed by multiple cell types during atherogenesis.10,15 In humans, it is believed to be atheroprotective, although the mechanism is not yet fully understood.14 In Apoe−/− mice, a model for atherosclerosis, the HSP70 levels correlated with lesion severity,15 suggesting that HSP70 expression may be upregulated for protection and thus be increased because of the greater disease burden. Levels of circulating HSP70 antibodies have also been correlated with the severity of vascular disease and may have prognostic value.10 It is possible that the relative levels of HSP70 and anti-HSP70 antibodies determines the HSP70 effect.
Increased expression of IL-6, an inflammatory cytokine, is associated with atherosclerosis and other vascular disease,16 and IL-6 addition enhances vascular calcification in vitro.23 In our experiments, HSP70 mediated the stimulatory effect of IL-6 on mineralization, which was abolished by HSP70 depletion. These results are more consistent with HSP70 being an inflammatory mediator with the potential to accelerate vascular disease rather than having a protective effect. However, it is also possible that our in vitro model does not accurately reflect the in vivo situation. Overall, the importance of the balance between BMP and BMP inhibitors, HSP70- and anti-HSP70 antibodies, and IL-6 and HSP70 in a given location makes it difficult to predict whether the ultimate effect will be pro- or antiatherogenic for individual cells and for the organism, as a whole.
Our results showed expression of HSP70, BMP-4, MGP, and IL-6 in proximity of the endothelium and in atherosclerotic lesions in Apoe−/− mice, which is consistent with previous reports3,4,15 and makes protein interactions plausible. Because inhibition of both BMP-4 and HSP70 decreased the procalcific effect of sera from Apoe−/− mice in vitro, circulating BMP-4 and HSP70 may affect inflammatory reactions and vascular calcification in the vascular wall. We were unable to test the effect of serum MGP in our study because of lack of neutralizing antibodies. However, circulating MGP has been reported to have very little effect on vascular calcification.29
Together, our results suggest that HSP70 binds MGP and enhances BMP activity, thereby functioning as a potential link between cellular stress, inflammation and BMP signaling. However, whether this effect in the end is beneficial or detrimental may depend on the particular context.
Sources of Funding
This work was supported part by NIH grants HL30568 and HL81397 and the American Heart Association (Western Affiliate).
This manuscript was sent to Donald D. Heistad, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received June 4, 2009; revision received July 23, 2009; accepted July 28, 2009.
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