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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

Bcr interaction with PDZK1 blunts VSM cell proliferation.A. VSM cells from PDZK1+/+ or PDZK1-/- mice were transfected with control dsRNA (Con siRNA) or dsRNA targeting Bcr (Bcr siRNA), and cell proliferation in response to PDGF-BB (10 ng/ml) was assessed by quantification of 3H-thymidine incorporation. Effective knockdown of Bcr is shown in Fig 4A. PDGF-BB-stimulated proliferation is expressed relative to proliferation in control, PBS-treated cells. Values are mean±SEM, n = 8, *p<0.05 vs. PDZK1+/+, †p<0.05 vs. Con siRNA. B. VSM cells were infected with adenovirus expressing vector alone (sham), wild-type Bcr or Bcr-V1271A, and relative Bcr expression was evaluated by immunoblot analysis. C. In cells described in B, proliferation was assessed in control PBS-treated cells (open bar) and in response to PDGF-BB (10 ng/ml, closed bar). PDGF-BB-stimulated proliferation is expressed relative to proliferation in control-treated cells. Values are mean±SEM, n = 3, *p<0.05 vs. no PDGF, †p<0.05 vs. wild-type Bcr.
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pone.0124494.g005: Bcr interaction with PDZK1 blunts VSM cell proliferation.A. VSM cells from PDZK1+/+ or PDZK1-/- mice were transfected with control dsRNA (Con siRNA) or dsRNA targeting Bcr (Bcr siRNA), and cell proliferation in response to PDGF-BB (10 ng/ml) was assessed by quantification of 3H-thymidine incorporation. Effective knockdown of Bcr is shown in Fig 4A. PDGF-BB-stimulated proliferation is expressed relative to proliferation in control, PBS-treated cells. Values are mean±SEM, n = 8, *p<0.05 vs. PDZK1+/+, †p<0.05 vs. Con siRNA. B. VSM cells were infected with adenovirus expressing vector alone (sham), wild-type Bcr or Bcr-V1271A, and relative Bcr expression was evaluated by immunoblot analysis. C. In cells described in B, proliferation was assessed in control PBS-treated cells (open bar) and in response to PDGF-BB (10 ng/ml, closed bar). PDGF-BB-stimulated proliferation is expressed relative to proliferation in control-treated cells. Values are mean±SEM, n = 3, *p<0.05 vs. no PDGF, †p<0.05 vs. wild-type Bcr.

Mentions: It was next determined whether Bcr is required for the exaggerated proliferation observed in response to PDGF in PDZK1-/- VSM cells using siRNA knockdown of the kinase. In siRNA control-transfected cells, PDZK1-/- cells showed exaggerated proliferation in response to PDGF (10 ng/ml) versus PDZK1+/+ cells (Fig 5A), confirming the results shown in Fig 3E. However, PDGF-stimulated proliferation was comparable in PDZK1+/+ versus PDZK1-/- cells depleted of Bcr. To determine how interaction with PDZK1 affects Bcr function in VSM, wild-type VSM cells were transfected with sham vector, cDNA encoding wild-type Bcr, or cDNA encoding a mutant form of Bcr with a disrupted C-terminal PDZ binding motif (Bcr-V1271A) that diminishes the interaction between Bcr and PDZK1 [20](Fig 5B). Compared to wild-type Bcr overexpression, the introduction of the Bcr-V1271A mutant yielded an exaggerated proliferation response to PDGF (Fig 5C). These findings indicate that Bcr protein is required for the exaggerated proliferation observed in PDZK1-/- VSM cells, and that the disruption of Bcr-PDZK1 interaction enhances VSM cell proliferation with PDGF. As such, PDZK1 tempers VSM cell proliferation via an inhibitory interaction with Bcr, revealing a mechanism whereby PDZK1 may afford protection from neointima formation.


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)

Bcr interaction with PDZK1 blunts VSM cell proliferation.A. VSM cells from PDZK1+/+ or PDZK1-/- mice were transfected with control dsRNA (Con siRNA) or dsRNA targeting Bcr (Bcr siRNA), and cell proliferation in response to PDGF-BB (10 ng/ml) was assessed by quantification of 3H-thymidine incorporation. Effective knockdown of Bcr is shown in Fig 4A. PDGF-BB-stimulated proliferation is expressed relative to proliferation in control, PBS-treated cells. Values are mean±SEM, n = 8, *p<0.05 vs. PDZK1+/+, †p<0.05 vs. Con siRNA. B. VSM cells were infected with adenovirus expressing vector alone (sham), wild-type Bcr or Bcr-V1271A, and relative Bcr expression was evaluated by immunoblot analysis. C. In cells described in B, proliferation was assessed in control PBS-treated cells (open bar) and in response to PDGF-BB (10 ng/ml, closed bar). PDGF-BB-stimulated proliferation is expressed relative to proliferation in control-treated cells. Values are mean±SEM, n = 3, *p<0.05 vs. no PDGF, †p<0.05 vs. wild-type Bcr.
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pone.0124494.g005: Bcr interaction with PDZK1 blunts VSM cell proliferation.A. VSM cells from PDZK1+/+ or PDZK1-/- mice were transfected with control dsRNA (Con siRNA) or dsRNA targeting Bcr (Bcr siRNA), and cell proliferation in response to PDGF-BB (10 ng/ml) was assessed by quantification of 3H-thymidine incorporation. Effective knockdown of Bcr is shown in Fig 4A. PDGF-BB-stimulated proliferation is expressed relative to proliferation in control, PBS-treated cells. Values are mean±SEM, n = 8, *p<0.05 vs. PDZK1+/+, †p<0.05 vs. Con siRNA. B. VSM cells were infected with adenovirus expressing vector alone (sham), wild-type Bcr or Bcr-V1271A, and relative Bcr expression was evaluated by immunoblot analysis. C. In cells described in B, proliferation was assessed in control PBS-treated cells (open bar) and in response to PDGF-BB (10 ng/ml, closed bar). PDGF-BB-stimulated proliferation is expressed relative to proliferation in control-treated cells. Values are mean±SEM, n = 3, *p<0.05 vs. no PDGF, †p<0.05 vs. wild-type Bcr.
Mentions: It was next determined whether Bcr is required for the exaggerated proliferation observed in response to PDGF in PDZK1-/- VSM cells using siRNA knockdown of the kinase. In siRNA control-transfected cells, PDZK1-/- cells showed exaggerated proliferation in response to PDGF (10 ng/ml) versus PDZK1+/+ cells (Fig 5A), confirming the results shown in Fig 3E. However, PDGF-stimulated proliferation was comparable in PDZK1+/+ versus PDZK1-/- cells depleted of Bcr. To determine how interaction with PDZK1 affects Bcr function in VSM, wild-type VSM cells were transfected with sham vector, cDNA encoding wild-type Bcr, or cDNA encoding a mutant form of Bcr with a disrupted C-terminal PDZ binding motif (Bcr-V1271A) that diminishes the interaction between Bcr and PDZK1 [20](Fig 5B). Compared to wild-type Bcr overexpression, the introduction of the Bcr-V1271A mutant yielded an exaggerated proliferation response to PDGF (Fig 5C). These findings indicate that Bcr protein is required for the exaggerated proliferation observed in PDZK1-/- VSM cells, and that the disruption of Bcr-PDZK1 interaction enhances VSM cell proliferation with PDGF. As such, PDZK1 tempers VSM cell proliferation via an inhibitory interaction with Bcr, revealing a mechanism whereby PDZK1 may afford protection from neointima formation.

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