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PDZK1 prevents neointima formation via suppression of breakpoint cluster region kinase in vascular smooth muscle.

Lee WR, Sacharidou A, Behling-Kelly E, Oltmann SC, Zhu W, Ahmed M, Gerard RD, Hui DY, Abe J, Shaul PW, Mineo C - PLoS ONE (2015)

Bottom Line: Tandem affinity purification-mass spectrometry revealed that PDZK1 interacts with breakpoint cluster region kinase (Bcr), which contains a C-terminal PDZ binding sequence and is known to enhance responses to PDGF in VSM.PDZK1 interaction with Bcr in VSM was demonstrated by pull-down and by coimmunoprecipitation, and the augmented proliferative response to PDGF in PDZK1-/- VSM was abrogated by Bcr depletion.Furthermore, compared with wild-type Bcr overexpression, the introduction of a Bcr mutant incapable of PDZK1 binding into VSM cells yielded an exaggerated proliferative response to PDGF.

View Article: PubMed Central - PubMed

Affiliation: Center for Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

ABSTRACT
Scavenger receptor class B, type I (SR-BI) and its adaptor protein PDZK1 mediate responses to HDL cholesterol in endothelium. Whether the receptor-adaptor protein tandem serves functions in other vascular cell types is unknown. The current work determined the roles of SR-BI and PDZK1 in vascular smooth muscle (VSM). To evaluate possible VSM functions of SR-BI and PDZK1 in vivo, neointima formation was assessed 21 days post-ligation in the carotid arteries of wild-type, SR-BI-/- or PDZK1-/- mice. Whereas neointima development was negligible in wild-type and SR-BI-/-, there was marked neointima formation in PDZK1-/- mice. PDZK1 expression was demonstrated in primary mouse VSM cells, and compared to wild-type cells, PDZK1-/- VSM displayed exaggerated proliferation and migration in response to platelet derived growth factor (PDGF). Tandem affinity purification-mass spectrometry revealed that PDZK1 interacts with breakpoint cluster region kinase (Bcr), which contains a C-terminal PDZ binding sequence and is known to enhance responses to PDGF in VSM. PDZK1 interaction with Bcr in VSM was demonstrated by pull-down and by coimmunoprecipitation, and the augmented proliferative response to PDGF in PDZK1-/- VSM was abrogated by Bcr depletion. Furthermore, compared with wild-type Bcr overexpression, the introduction of a Bcr mutant incapable of PDZK1 binding into VSM cells yielded an exaggerated proliferative response to PDGF. Thus, PDZK1 has novel SR-BI-independent function in VSM that affords protection from neointima formation, and this involves PDZK1 suppression of VSM cell proliferation via an inhibitory interaction with Bcr.

No MeSH data available.


Related in: MedlinePlus

VSM cells from PDZK1-/- mice display enhanced PDGF-stimulated cell proliferation and migration.A. Aortic VSM cells were isolated from PDZK1+/+ or PDZK1-/- mice and immmunfluorescent staining was performed with anti-α-SMA monoclonal antibody (red). The scale bar indicates 100 μm. B. PDZK1 mRNA was quantified in VSM cells and liver from PDZK1+/+ and PDZK1-/- mice. Results are expressed relative to cyclophilin. C. PDZK1 protein expression was evaluated by immunoblotting using anti-PDZK1 polyclonal antibody. Equal protein loading was confirmed by immunoblotting using anti-GAPDH antibody. D. Cell growth under quiescent conditions was assessed by quantification of 3H-thymidine incorporation. E.Cell proliferation was evaluated in VSM incubated in the presence of 0–20 ng/ml PDGF-BB for 24h. F. Cell migration was assessed in VSM incubated in the presence of 0–30 ng/ml PDGF-BB for 4h. In B and D-F, values are mean±SEM, n = 3, *p<0.05 vs. no PDGF, †p<0.05 vs. PDZK1+/+.
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pone.0124494.g003: VSM cells from PDZK1-/- mice display enhanced PDGF-stimulated cell proliferation and migration.A. Aortic VSM cells were isolated from PDZK1+/+ or PDZK1-/- mice and immmunfluorescent staining was performed with anti-α-SMA monoclonal antibody (red). The scale bar indicates 100 μm. B. PDZK1 mRNA was quantified in VSM cells and liver from PDZK1+/+ and PDZK1-/- mice. Results are expressed relative to cyclophilin. C. PDZK1 protein expression was evaluated by immunoblotting using anti-PDZK1 polyclonal antibody. Equal protein loading was confirmed by immunoblotting using anti-GAPDH antibody. D. Cell growth under quiescent conditions was assessed by quantification of 3H-thymidine incorporation. E.Cell proliferation was evaluated in VSM incubated in the presence of 0–20 ng/ml PDGF-BB for 24h. F. Cell migration was assessed in VSM incubated in the presence of 0–30 ng/ml PDGF-BB for 4h. In B and D-F, values are mean±SEM, n = 3, *p<0.05 vs. no PDGF, †p<0.05 vs. PDZK1+/+.

Mentions: To directly determine if PDZK1 influences the proliferation or migration of VSM cells, primary VSM cells were isolated from the aortas of wild-type PDZK1+/+ and PDZK1-/- mice by enzymatic dispersion [24]. The early passage VSM cells from the two genotype groups were indistinguishable, displaying a typical spindle shape and filamentous αSMA bundles spanning the length of the cells, and more than 90% of cells displayed positive immunohistochemical staining for αSMA (Fig 3A). PDZK1 expression in VSM cells was then evaluated by RT-PCR and immunoblotting, using liver as a positive control tissue (Fig 3B and 3C); PDZK1 transcript and protein were detectable in PDZK1+/+ VSM and they were predictably absent in PDZK1-/- cells. To assess the relative expression of PDZK1 in VSM, parallel quantification of the transcript was done in liver where PDZK1 expression is substantial [25]. The abundance of PDZK1 mRNA in VSM was less than 0.2% of that found in the liver.


PDZK1 prevents neointima formation via suppression of breakpoint cluster region kinase in vascular smooth muscle.

Lee WR, Sacharidou A, Behling-Kelly E, Oltmann SC, Zhu W, Ahmed M, Gerard RD, Hui DY, Abe J, Shaul PW, Mineo C - PLoS ONE (2015)

VSM cells from PDZK1-/- mice display enhanced PDGF-stimulated cell proliferation and migration.A. Aortic VSM cells were isolated from PDZK1+/+ or PDZK1-/- mice and immmunfluorescent staining was performed with anti-α-SMA monoclonal antibody (red). The scale bar indicates 100 μm. B. PDZK1 mRNA was quantified in VSM cells and liver from PDZK1+/+ and PDZK1-/- mice. Results are expressed relative to cyclophilin. C. PDZK1 protein expression was evaluated by immunoblotting using anti-PDZK1 polyclonal antibody. Equal protein loading was confirmed by immunoblotting using anti-GAPDH antibody. D. Cell growth under quiescent conditions was assessed by quantification of 3H-thymidine incorporation. E.Cell proliferation was evaluated in VSM incubated in the presence of 0–20 ng/ml PDGF-BB for 24h. F. Cell migration was assessed in VSM incubated in the presence of 0–30 ng/ml PDGF-BB for 4h. In B and D-F, values are mean±SEM, n = 3, *p<0.05 vs. no PDGF, †p<0.05 vs. PDZK1+/+.
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pone.0124494.g003: VSM cells from PDZK1-/- mice display enhanced PDGF-stimulated cell proliferation and migration.A. Aortic VSM cells were isolated from PDZK1+/+ or PDZK1-/- mice and immmunfluorescent staining was performed with anti-α-SMA monoclonal antibody (red). The scale bar indicates 100 μm. B. PDZK1 mRNA was quantified in VSM cells and liver from PDZK1+/+ and PDZK1-/- mice. Results are expressed relative to cyclophilin. C. PDZK1 protein expression was evaluated by immunoblotting using anti-PDZK1 polyclonal antibody. Equal protein loading was confirmed by immunoblotting using anti-GAPDH antibody. D. Cell growth under quiescent conditions was assessed by quantification of 3H-thymidine incorporation. E.Cell proliferation was evaluated in VSM incubated in the presence of 0–20 ng/ml PDGF-BB for 24h. F. Cell migration was assessed in VSM incubated in the presence of 0–30 ng/ml PDGF-BB for 4h. In B and D-F, values are mean±SEM, n = 3, *p<0.05 vs. no PDGF, †p<0.05 vs. PDZK1+/+.
Mentions: To directly determine if PDZK1 influences the proliferation or migration of VSM cells, primary VSM cells were isolated from the aortas of wild-type PDZK1+/+ and PDZK1-/- mice by enzymatic dispersion [24]. The early passage VSM cells from the two genotype groups were indistinguishable, displaying a typical spindle shape and filamentous αSMA bundles spanning the length of the cells, and more than 90% of cells displayed positive immunohistochemical staining for αSMA (Fig 3A). PDZK1 expression in VSM cells was then evaluated by RT-PCR and immunoblotting, using liver as a positive control tissue (Fig 3B and 3C); PDZK1 transcript and protein were detectable in PDZK1+/+ VSM and they were predictably absent in PDZK1-/- cells. To assess the relative expression of PDZK1 in VSM, parallel quantification of the transcript was done in liver where PDZK1 expression is substantial [25]. The abundance of PDZK1 mRNA in VSM was less than 0.2% of that found in the liver.

Bottom Line: Tandem affinity purification-mass spectrometry revealed that PDZK1 interacts with breakpoint cluster region kinase (Bcr), which contains a C-terminal PDZ binding sequence and is known to enhance responses to PDGF in VSM.PDZK1 interaction with Bcr in VSM was demonstrated by pull-down and by coimmunoprecipitation, and the augmented proliferative response to PDGF in PDZK1-/- VSM was abrogated by Bcr depletion.Furthermore, compared with wild-type Bcr overexpression, the introduction of a Bcr mutant incapable of PDZK1 binding into VSM cells yielded an exaggerated proliferative response to PDGF.

View Article: PubMed Central - PubMed

Affiliation: Center for Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

ABSTRACT
Scavenger receptor class B, type I (SR-BI) and its adaptor protein PDZK1 mediate responses to HDL cholesterol in endothelium. Whether the receptor-adaptor protein tandem serves functions in other vascular cell types is unknown. The current work determined the roles of SR-BI and PDZK1 in vascular smooth muscle (VSM). To evaluate possible VSM functions of SR-BI and PDZK1 in vivo, neointima formation was assessed 21 days post-ligation in the carotid arteries of wild-type, SR-BI-/- or PDZK1-/- mice. Whereas neointima development was negligible in wild-type and SR-BI-/-, there was marked neointima formation in PDZK1-/- mice. PDZK1 expression was demonstrated in primary mouse VSM cells, and compared to wild-type cells, PDZK1-/- VSM displayed exaggerated proliferation and migration in response to platelet derived growth factor (PDGF). Tandem affinity purification-mass spectrometry revealed that PDZK1 interacts with breakpoint cluster region kinase (Bcr), which contains a C-terminal PDZ binding sequence and is known to enhance responses to PDGF in VSM. PDZK1 interaction with Bcr in VSM was demonstrated by pull-down and by coimmunoprecipitation, and the augmented proliferative response to PDGF in PDZK1-/- VSM was abrogated by Bcr depletion. Furthermore, compared with wild-type Bcr overexpression, the introduction of a Bcr mutant incapable of PDZK1 binding into VSM cells yielded an exaggerated proliferative response to PDGF. Thus, PDZK1 has novel SR-BI-independent function in VSM that affords protection from neointima formation, and this involves PDZK1 suppression of VSM cell proliferation via an inhibitory interaction with Bcr.

No MeSH data available.


Related in: MedlinePlus