Limits...
Vascular Proteomics Reveal Novel Proteins Involved in SMC Phenotypic Change: OLR1 as a SMC Receptor Regulating Proliferation and Inflammatory Response.

Kang DH, Choi M, Chang S, Lee MY, Lee DJ, Choi K, Park J, Han EC, Hwang D, Kwon K, Jo H, Choi C, Kang SW - PLoS ONE (2015)

Bottom Line: The differential proteomics analysis in a balloon-induced injury model of rat carotid artery revealed that the expressions of 44 proteins are changed within 3 days post injury.Importantly, OLR1 and PDGFRβ were associated in close proximity in the plasma membrane.Thus, this study elicits the protein network organizing the phenotypic change of VSMC in the vascular injury diseases such as atherosclerosis and discovers OLR1 as a novel molecular link between the proliferative and inflammatory responses of VSMCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science and Research Center for Cell Homeostasis, Ewha Womans University, Seoul 120-750, Korea.

ABSTRACT
Neointimal hyperplasia of vascular smooth muscle cells (VSMC) plays a critical role in atherosclerotic plaque formation and in-stent restenosis, but the underlying mechanisms are still incompletely understood. We performed a proteomics study to identify novel signaling molecules organizing the VSMC hyperplasia. The differential proteomics analysis in a balloon-induced injury model of rat carotid artery revealed that the expressions of 44 proteins are changed within 3 days post injury. The combination of cellular function assays and a protein network analysis further demonstrated that 27 out of 44 proteins constitute key signaling networks orchestrating the phenotypic change of VSMC from contractile to epithelial-like synthetic. Among the list of proteins, the in vivo validation specifically revealed that six proteins (Rab15, ITR, OLR1, PDHβ, PTPε) are positive regulators for VSMC hyperplasia. In particular, the OLR1 played dual roles in the VSMC hyperplasia by directly mediating oxidized LDL-induced monocyte adhesion via NF-κB activation and by assisting the PDGF-induced proliferation/migration. Importantly, OLR1 and PDGFRβ were associated in close proximity in the plasma membrane. Thus, this study elicits the protein network organizing the phenotypic change of VSMC in the vascular injury diseases such as atherosclerosis and discovers OLR1 as a novel molecular link between the proliferative and inflammatory responses of VSMCs.

No MeSH data available.


Related in: MedlinePlus

OLR1 is associated with the PDGF signaling in VSMCs.(A) PDGF-dependent tyrosine phosphorylation in VSMCs transfected with control or OLR1 siRNA. The HASMCs were starved in serum-free culture media for 18 hr and the stimulated with PDGF-BB (25 ng/ml) for the indicated times. The protein tyrosine phosphorylation was detected using anti-phosphotyrosine antibody (4G10) and the PDGFR activation was measured by immunoblotting the Tyr857 phosphorylation on PDGFRβ. A representative immunoblot is shown (n = 3). (B—D) Effect of OxLDL treatment on PDGF-induced tyrosine phosphorylation in VSMCs. The serum-starved VSMCs were co-treated with PDGF-BB and OxLDL (B) or pretreated with OxLDL for 1 hr (C) and 24 hr (D) before PDGF-BB treatment. The activation of MAP kinase ERK was measured by phospho-specific antibody (pTpY). A representative immunoblot is shown (n = 3). (E) Subcellular distribution of OLR1 in OxLDL-treated VSMCs. Cells were treated with OxLDL (100 μg/ml) for 1 hr or 24 hr and the cell extracts were then separated to the subcellular fractions by differential centrifugation. A representative immunoblot is shown (n = 3). (F) The proximity ligation assay showing the OLR1 and PDGFRβ interaction in the plasma membrane of VSMCs. The proximity ligation assay was performed using DuoLink in situ probes with the indicated antibodies. DIC and DAPI-stained images are merged with DuoLink fluorescence image (merge). Representative images from two experiments are shown. (G) OLR1 plays a dual receptor function in VSMCs. OLR1 is expressed in VSMCs and also induced in the arterial vessels by an endothelial denudation. OLR1 primarily functions as an oxidized LDL receptor in VSMCs that mediates the NF-κB-dependent inflammatory response. OLR1 itself enhances the PDGF-induced signal transduction including PDGFRβ and ERK phosphorylation. Therefore, the OLR1 is the first SMC receptor that mediates the pro-inflammatory and proliferative responses in a ligand-dependent and -independent manner, respectively. Purple colored box is a leucine repeat in OLR1.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4548952&req=5

pone.0133845.g007: OLR1 is associated with the PDGF signaling in VSMCs.(A) PDGF-dependent tyrosine phosphorylation in VSMCs transfected with control or OLR1 siRNA. The HASMCs were starved in serum-free culture media for 18 hr and the stimulated with PDGF-BB (25 ng/ml) for the indicated times. The protein tyrosine phosphorylation was detected using anti-phosphotyrosine antibody (4G10) and the PDGFR activation was measured by immunoblotting the Tyr857 phosphorylation on PDGFRβ. A representative immunoblot is shown (n = 3). (B—D) Effect of OxLDL treatment on PDGF-induced tyrosine phosphorylation in VSMCs. The serum-starved VSMCs were co-treated with PDGF-BB and OxLDL (B) or pretreated with OxLDL for 1 hr (C) and 24 hr (D) before PDGF-BB treatment. The activation of MAP kinase ERK was measured by phospho-specific antibody (pTpY). A representative immunoblot is shown (n = 3). (E) Subcellular distribution of OLR1 in OxLDL-treated VSMCs. Cells were treated with OxLDL (100 μg/ml) for 1 hr or 24 hr and the cell extracts were then separated to the subcellular fractions by differential centrifugation. A representative immunoblot is shown (n = 3). (F) The proximity ligation assay showing the OLR1 and PDGFRβ interaction in the plasma membrane of VSMCs. The proximity ligation assay was performed using DuoLink in situ probes with the indicated antibodies. DIC and DAPI-stained images are merged with DuoLink fluorescence image (merge). Representative images from two experiments are shown. (G) OLR1 plays a dual receptor function in VSMCs. OLR1 is expressed in VSMCs and also induced in the arterial vessels by an endothelial denudation. OLR1 primarily functions as an oxidized LDL receptor in VSMCs that mediates the NF-κB-dependent inflammatory response. OLR1 itself enhances the PDGF-induced signal transduction including PDGFRβ and ERK phosphorylation. Therefore, the OLR1 is the first SMC receptor that mediates the pro-inflammatory and proliferative responses in a ligand-dependent and -independent manner, respectively. Purple colored box is a leucine repeat in OLR1.

Mentions: Since OLR1 played a role in PDGF-induced proliferation and migration, we further examined the effect of the OLR1 on PDGF signaling. The knockdown of OLR1 expression decreased the protein tyrosine phosphorylation including PDGFRβ phosphorylation in response to PDGF stimulation (Fig 7A). We then hypothesized that OLR1 would function as a co-receptor for the full activation of PDGFRβ in VSMCs. To test this hypothesis, we explored whether there is a functional interaction between PDGFRβ and OLR1 in VSMCs. The mmLDL treatment neither induced the PDGFRβ phosphorylation in VSMCs nor interfered with the PDGF-induced tyrosine phosphorylation (Fig 7B). This result indicates that the mmLDL-OLR1 interaction was PDGFRβ-independent. In addition, a slight activation of tyrosine phosphorylation and ERK kinase by mmLDL alone might be due partly to the formation of hydroxynonenal-PDGFR adduct [28]. However, the 1-hr pretreatment of mmLDL significantly reduced the PDGF-induced tyrosine phosphorylation (Fig 7C). In contrast, the 24-hr pretreatment of mmLDL at various doses had no effect on the PDGF signaling (Fig 7D). It is known that the scavenger receptors like OLR1 are endocytosed after the ligand engagement [29, 30]. Indeed, the OLR1 was endocytosed from membrane to cytosolic fraction by 1-hr treatment of mmLDLs, whereas it was completely recovered in the membrane after 24 hr (Fig 7E). To further identify the interaction between OLR1 and PDGFRβ, we performed the proximity ligation assay using DuoLink. The combination of anti-PDGFRβ and anti-OLR1 antibodies, not the anti-PDGFRβ antibody alone, showed that OLR1 interacted with PDGFRβ in close proximity in the plasma membrane of VSMCs (Fig 7F). Together, these results suggest that the mmLDL-dependent clearance or genetic depletion of OLR1 from the plasma membrane may dampen the PDGF-PDGFRβ signaling events in VSMCs.


Vascular Proteomics Reveal Novel Proteins Involved in SMC Phenotypic Change: OLR1 as a SMC Receptor Regulating Proliferation and Inflammatory Response.

Kang DH, Choi M, Chang S, Lee MY, Lee DJ, Choi K, Park J, Han EC, Hwang D, Kwon K, Jo H, Choi C, Kang SW - PLoS ONE (2015)

OLR1 is associated with the PDGF signaling in VSMCs.(A) PDGF-dependent tyrosine phosphorylation in VSMCs transfected with control or OLR1 siRNA. The HASMCs were starved in serum-free culture media for 18 hr and the stimulated with PDGF-BB (25 ng/ml) for the indicated times. The protein tyrosine phosphorylation was detected using anti-phosphotyrosine antibody (4G10) and the PDGFR activation was measured by immunoblotting the Tyr857 phosphorylation on PDGFRβ. A representative immunoblot is shown (n = 3). (B—D) Effect of OxLDL treatment on PDGF-induced tyrosine phosphorylation in VSMCs. The serum-starved VSMCs were co-treated with PDGF-BB and OxLDL (B) or pretreated with OxLDL for 1 hr (C) and 24 hr (D) before PDGF-BB treatment. The activation of MAP kinase ERK was measured by phospho-specific antibody (pTpY). A representative immunoblot is shown (n = 3). (E) Subcellular distribution of OLR1 in OxLDL-treated VSMCs. Cells were treated with OxLDL (100 μg/ml) for 1 hr or 24 hr and the cell extracts were then separated to the subcellular fractions by differential centrifugation. A representative immunoblot is shown (n = 3). (F) The proximity ligation assay showing the OLR1 and PDGFRβ interaction in the plasma membrane of VSMCs. The proximity ligation assay was performed using DuoLink in situ probes with the indicated antibodies. DIC and DAPI-stained images are merged with DuoLink fluorescence image (merge). Representative images from two experiments are shown. (G) OLR1 plays a dual receptor function in VSMCs. OLR1 is expressed in VSMCs and also induced in the arterial vessels by an endothelial denudation. OLR1 primarily functions as an oxidized LDL receptor in VSMCs that mediates the NF-κB-dependent inflammatory response. OLR1 itself enhances the PDGF-induced signal transduction including PDGFRβ and ERK phosphorylation. Therefore, the OLR1 is the first SMC receptor that mediates the pro-inflammatory and proliferative responses in a ligand-dependent and -independent manner, respectively. Purple colored box is a leucine repeat in OLR1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4548952&req=5

pone.0133845.g007: OLR1 is associated with the PDGF signaling in VSMCs.(A) PDGF-dependent tyrosine phosphorylation in VSMCs transfected with control or OLR1 siRNA. The HASMCs were starved in serum-free culture media for 18 hr and the stimulated with PDGF-BB (25 ng/ml) for the indicated times. The protein tyrosine phosphorylation was detected using anti-phosphotyrosine antibody (4G10) and the PDGFR activation was measured by immunoblotting the Tyr857 phosphorylation on PDGFRβ. A representative immunoblot is shown (n = 3). (B—D) Effect of OxLDL treatment on PDGF-induced tyrosine phosphorylation in VSMCs. The serum-starved VSMCs were co-treated with PDGF-BB and OxLDL (B) or pretreated with OxLDL for 1 hr (C) and 24 hr (D) before PDGF-BB treatment. The activation of MAP kinase ERK was measured by phospho-specific antibody (pTpY). A representative immunoblot is shown (n = 3). (E) Subcellular distribution of OLR1 in OxLDL-treated VSMCs. Cells were treated with OxLDL (100 μg/ml) for 1 hr or 24 hr and the cell extracts were then separated to the subcellular fractions by differential centrifugation. A representative immunoblot is shown (n = 3). (F) The proximity ligation assay showing the OLR1 and PDGFRβ interaction in the plasma membrane of VSMCs. The proximity ligation assay was performed using DuoLink in situ probes with the indicated antibodies. DIC and DAPI-stained images are merged with DuoLink fluorescence image (merge). Representative images from two experiments are shown. (G) OLR1 plays a dual receptor function in VSMCs. OLR1 is expressed in VSMCs and also induced in the arterial vessels by an endothelial denudation. OLR1 primarily functions as an oxidized LDL receptor in VSMCs that mediates the NF-κB-dependent inflammatory response. OLR1 itself enhances the PDGF-induced signal transduction including PDGFRβ and ERK phosphorylation. Therefore, the OLR1 is the first SMC receptor that mediates the pro-inflammatory and proliferative responses in a ligand-dependent and -independent manner, respectively. Purple colored box is a leucine repeat in OLR1.
Mentions: Since OLR1 played a role in PDGF-induced proliferation and migration, we further examined the effect of the OLR1 on PDGF signaling. The knockdown of OLR1 expression decreased the protein tyrosine phosphorylation including PDGFRβ phosphorylation in response to PDGF stimulation (Fig 7A). We then hypothesized that OLR1 would function as a co-receptor for the full activation of PDGFRβ in VSMCs. To test this hypothesis, we explored whether there is a functional interaction between PDGFRβ and OLR1 in VSMCs. The mmLDL treatment neither induced the PDGFRβ phosphorylation in VSMCs nor interfered with the PDGF-induced tyrosine phosphorylation (Fig 7B). This result indicates that the mmLDL-OLR1 interaction was PDGFRβ-independent. In addition, a slight activation of tyrosine phosphorylation and ERK kinase by mmLDL alone might be due partly to the formation of hydroxynonenal-PDGFR adduct [28]. However, the 1-hr pretreatment of mmLDL significantly reduced the PDGF-induced tyrosine phosphorylation (Fig 7C). In contrast, the 24-hr pretreatment of mmLDL at various doses had no effect on the PDGF signaling (Fig 7D). It is known that the scavenger receptors like OLR1 are endocytosed after the ligand engagement [29, 30]. Indeed, the OLR1 was endocytosed from membrane to cytosolic fraction by 1-hr treatment of mmLDLs, whereas it was completely recovered in the membrane after 24 hr (Fig 7E). To further identify the interaction between OLR1 and PDGFRβ, we performed the proximity ligation assay using DuoLink. The combination of anti-PDGFRβ and anti-OLR1 antibodies, not the anti-PDGFRβ antibody alone, showed that OLR1 interacted with PDGFRβ in close proximity in the plasma membrane of VSMCs (Fig 7F). Together, these results suggest that the mmLDL-dependent clearance or genetic depletion of OLR1 from the plasma membrane may dampen the PDGF-PDGFRβ signaling events in VSMCs.

Bottom Line: The differential proteomics analysis in a balloon-induced injury model of rat carotid artery revealed that the expressions of 44 proteins are changed within 3 days post injury.Importantly, OLR1 and PDGFRβ were associated in close proximity in the plasma membrane.Thus, this study elicits the protein network organizing the phenotypic change of VSMC in the vascular injury diseases such as atherosclerosis and discovers OLR1 as a novel molecular link between the proliferative and inflammatory responses of VSMCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science and Research Center for Cell Homeostasis, Ewha Womans University, Seoul 120-750, Korea.

ABSTRACT
Neointimal hyperplasia of vascular smooth muscle cells (VSMC) plays a critical role in atherosclerotic plaque formation and in-stent restenosis, but the underlying mechanisms are still incompletely understood. We performed a proteomics study to identify novel signaling molecules organizing the VSMC hyperplasia. The differential proteomics analysis in a balloon-induced injury model of rat carotid artery revealed that the expressions of 44 proteins are changed within 3 days post injury. The combination of cellular function assays and a protein network analysis further demonstrated that 27 out of 44 proteins constitute key signaling networks orchestrating the phenotypic change of VSMC from contractile to epithelial-like synthetic. Among the list of proteins, the in vivo validation specifically revealed that six proteins (Rab15, ITR, OLR1, PDHβ, PTPε) are positive regulators for VSMC hyperplasia. In particular, the OLR1 played dual roles in the VSMC hyperplasia by directly mediating oxidized LDL-induced monocyte adhesion via NF-κB activation and by assisting the PDGF-induced proliferation/migration. Importantly, OLR1 and PDGFRβ were associated in close proximity in the plasma membrane. Thus, this study elicits the protein network organizing the phenotypic change of VSMC in the vascular injury diseases such as atherosclerosis and discovers OLR1 as a novel molecular link between the proliferative and inflammatory responses of VSMCs.

No MeSH data available.


Related in: MedlinePlus