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F‑actin cytoskeleton reorganization is associated with hepatic stellate cell activation.

Cui X, Zhang X, Yin Q, Meng A, Su S, Jing X, Li H, Guan X, Li X, Liu S, Cheng M - Mol Med Rep (2014)

Bottom Line: Treatment with Jas resulted in thick actin bundles and a patchy appearance in the cytoplasm in HSC-T6 cells.Furthermore, the activation of HSC-T6 cells induced by the reorganization of the actin cytoskeleton was associated with the p38 mitogen-activated protein kinase (p38 MAPK) pathway.In conclusion, the present study suggests that the reorganization of the F-actin cytoskeleton is associated with HSC activation and that the p38 MAPK pathway is involved in this process.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Center, Weifang Medical University, Weifang, Shandong 261053, P.R. China.

ABSTRACT
The activation of hepatic stellate cells (HSCs) is involved in the development of hepatic fibrosis. Previous studies have indicated that the acquisition of certain properties by activated HSCs is highly dependent on the reorganization of the actin cytoskeleton. However, direct evidence showing that the reorganization of the actin cytoskeleton is responsible for HSC activation is lacking. The aim of the present study was to investigate the role of cytoskeletal reorganization during HSC activation and to clarify the underlying mechanism. HSC-T6 cells were treated either with the F-actin stabilizer jasplakinolide (Jas) or the depolymerizer cytochalasin D (Cyto D). The actin cytoskeleton was evaluated via assessment of stress fiber formation. Furthermore, the activation properties of HSCs, including proliferation, adhesion, migration and the expression of α-smooth muscle actin (α-SMA) and collagen 1, were investigated in vitro. The results showed that Jas and Cyto D affected the actin distribution in HSC-T6 cells. Treatment with Jas resulted in thick actin bundles and a patchy appearance in the cytoplasm in HSC-T6 cells. In parallel, polymerization of actin microfilaments induced by Jas upregulated the expression of α-SMA and collagen 1, and also enhanced the migration and adhesion properties of HSC-T6 cells. Furthermore, the activation of HSC-T6 cells induced by the reorganization of the actin cytoskeleton was associated with the p38 mitogen-activated protein kinase (p38 MAPK) pathway. In conclusion, the present study suggests that the reorganization of the F-actin cytoskeleton is associated with HSC activation and that the p38 MAPK pathway is involved in this process. The inhibition of F-actin reorganization may thus be a potential key factor or molecular target for the control of liver fibrosis or cirrhosis.

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Actin cytoskeleton reorganization-induced HSC-T6 cell activation correlates with the p38 MAPK pathway. (A) HSC-T6 cells were pretreated with PD98059 (10 μmol/l) or SB203580 (100 nmol/l), respectively, for 30 min. The cells were then exposed to DMSO or Jas. Gene expression of α-SMA and collagen type 1 was then determined using the quantitative polymerase chain reaction. (B) The phosphorylation status of p-38 MAPK was assessed by western blot analysis. HSC-T6 cells were incubated with DMSO or Jas for 1 h. Cell lysates were resolved using 12% SDS-PAGE, followed by transfer to a polyvinylidene fluoride membrane. Western blot analysis was performed with specific antibodies to distinguish between different phosphorylation statuses of p38 MAPK. In addition, total p38 MAPK was analyzed as a loading control. P-p38 MAPK was densitometrically analyzed and normalized to total p38 MAPK. The results are expressed as the mean ± standard error of five experiments. *P<0.05. α-SMA, α-smooth muscle actin; IOD, integrated optical density; Jas, jasplakinolide; DMSO, dimethylsulfoxide; p38 MAPK, p38 mitogen-activated protein kinase; P-p38, phosphorylated p38 MAPK.
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f4-mmr-09-05-1641: Actin cytoskeleton reorganization-induced HSC-T6 cell activation correlates with the p38 MAPK pathway. (A) HSC-T6 cells were pretreated with PD98059 (10 μmol/l) or SB203580 (100 nmol/l), respectively, for 30 min. The cells were then exposed to DMSO or Jas. Gene expression of α-SMA and collagen type 1 was then determined using the quantitative polymerase chain reaction. (B) The phosphorylation status of p-38 MAPK was assessed by western blot analysis. HSC-T6 cells were incubated with DMSO or Jas for 1 h. Cell lysates were resolved using 12% SDS-PAGE, followed by transfer to a polyvinylidene fluoride membrane. Western blot analysis was performed with specific antibodies to distinguish between different phosphorylation statuses of p38 MAPK. In addition, total p38 MAPK was analyzed as a loading control. P-p38 MAPK was densitometrically analyzed and normalized to total p38 MAPK. The results are expressed as the mean ± standard error of five experiments. *P<0.05. α-SMA, α-smooth muscle actin; IOD, integrated optical density; Jas, jasplakinolide; DMSO, dimethylsulfoxide; p38 MAPK, p38 mitogen-activated protein kinase; P-p38, phosphorylated p38 MAPK.

Mentions: Both extracellular signal-regulated kinase (ERK) and p38 MAPK have been shown to regulate HSC activation (14,15). To explore whether those signaling molecules were involved in the actin cytoskeleton reorganization-induced HSC activation, HSC-T6 cells were pre-incubated for 30 min with inhibitors of ERK (PD98059) or p38 MAPK (SB203580) prior to treatment with Jas. qPCR revealed that PD98059 did not affect the expression of α-SMA or collagen type 1 induced by Jas. By contrast, inhibition of p38 MAPK by SB203580 significantly reduced the Jas-induced expression of α-SMA and collagen type 1 (Fig. 4A).


F‑actin cytoskeleton reorganization is associated with hepatic stellate cell activation.

Cui X, Zhang X, Yin Q, Meng A, Su S, Jing X, Li H, Guan X, Li X, Liu S, Cheng M - Mol Med Rep (2014)

Actin cytoskeleton reorganization-induced HSC-T6 cell activation correlates with the p38 MAPK pathway. (A) HSC-T6 cells were pretreated with PD98059 (10 μmol/l) or SB203580 (100 nmol/l), respectively, for 30 min. The cells were then exposed to DMSO or Jas. Gene expression of α-SMA and collagen type 1 was then determined using the quantitative polymerase chain reaction. (B) The phosphorylation status of p-38 MAPK was assessed by western blot analysis. HSC-T6 cells were incubated with DMSO or Jas for 1 h. Cell lysates were resolved using 12% SDS-PAGE, followed by transfer to a polyvinylidene fluoride membrane. Western blot analysis was performed with specific antibodies to distinguish between different phosphorylation statuses of p38 MAPK. In addition, total p38 MAPK was analyzed as a loading control. P-p38 MAPK was densitometrically analyzed and normalized to total p38 MAPK. The results are expressed as the mean ± standard error of five experiments. *P<0.05. α-SMA, α-smooth muscle actin; IOD, integrated optical density; Jas, jasplakinolide; DMSO, dimethylsulfoxide; p38 MAPK, p38 mitogen-activated protein kinase; P-p38, phosphorylated p38 MAPK.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4020483&req=5

f4-mmr-09-05-1641: Actin cytoskeleton reorganization-induced HSC-T6 cell activation correlates with the p38 MAPK pathway. (A) HSC-T6 cells were pretreated with PD98059 (10 μmol/l) or SB203580 (100 nmol/l), respectively, for 30 min. The cells were then exposed to DMSO or Jas. Gene expression of α-SMA and collagen type 1 was then determined using the quantitative polymerase chain reaction. (B) The phosphorylation status of p-38 MAPK was assessed by western blot analysis. HSC-T6 cells were incubated with DMSO or Jas for 1 h. Cell lysates were resolved using 12% SDS-PAGE, followed by transfer to a polyvinylidene fluoride membrane. Western blot analysis was performed with specific antibodies to distinguish between different phosphorylation statuses of p38 MAPK. In addition, total p38 MAPK was analyzed as a loading control. P-p38 MAPK was densitometrically analyzed and normalized to total p38 MAPK. The results are expressed as the mean ± standard error of five experiments. *P<0.05. α-SMA, α-smooth muscle actin; IOD, integrated optical density; Jas, jasplakinolide; DMSO, dimethylsulfoxide; p38 MAPK, p38 mitogen-activated protein kinase; P-p38, phosphorylated p38 MAPK.
Mentions: Both extracellular signal-regulated kinase (ERK) and p38 MAPK have been shown to regulate HSC activation (14,15). To explore whether those signaling molecules were involved in the actin cytoskeleton reorganization-induced HSC activation, HSC-T6 cells were pre-incubated for 30 min with inhibitors of ERK (PD98059) or p38 MAPK (SB203580) prior to treatment with Jas. qPCR revealed that PD98059 did not affect the expression of α-SMA or collagen type 1 induced by Jas. By contrast, inhibition of p38 MAPK by SB203580 significantly reduced the Jas-induced expression of α-SMA and collagen type 1 (Fig. 4A).

Bottom Line: Treatment with Jas resulted in thick actin bundles and a patchy appearance in the cytoplasm in HSC-T6 cells.Furthermore, the activation of HSC-T6 cells induced by the reorganization of the actin cytoskeleton was associated with the p38 mitogen-activated protein kinase (p38 MAPK) pathway.In conclusion, the present study suggests that the reorganization of the F-actin cytoskeleton is associated with HSC activation and that the p38 MAPK pathway is involved in this process.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Center, Weifang Medical University, Weifang, Shandong 261053, P.R. China.

ABSTRACT
The activation of hepatic stellate cells (HSCs) is involved in the development of hepatic fibrosis. Previous studies have indicated that the acquisition of certain properties by activated HSCs is highly dependent on the reorganization of the actin cytoskeleton. However, direct evidence showing that the reorganization of the actin cytoskeleton is responsible for HSC activation is lacking. The aim of the present study was to investigate the role of cytoskeletal reorganization during HSC activation and to clarify the underlying mechanism. HSC-T6 cells were treated either with the F-actin stabilizer jasplakinolide (Jas) or the depolymerizer cytochalasin D (Cyto D). The actin cytoskeleton was evaluated via assessment of stress fiber formation. Furthermore, the activation properties of HSCs, including proliferation, adhesion, migration and the expression of α-smooth muscle actin (α-SMA) and collagen 1, were investigated in vitro. The results showed that Jas and Cyto D affected the actin distribution in HSC-T6 cells. Treatment with Jas resulted in thick actin bundles and a patchy appearance in the cytoplasm in HSC-T6 cells. In parallel, polymerization of actin microfilaments induced by Jas upregulated the expression of α-SMA and collagen 1, and also enhanced the migration and adhesion properties of HSC-T6 cells. Furthermore, the activation of HSC-T6 cells induced by the reorganization of the actin cytoskeleton was associated with the p38 mitogen-activated protein kinase (p38 MAPK) pathway. In conclusion, the present study suggests that the reorganization of the F-actin cytoskeleton is associated with HSC activation and that the p38 MAPK pathway is involved in this process. The inhibition of F-actin reorganization may thus be a potential key factor or molecular target for the control of liver fibrosis or cirrhosis.

Show MeSH
Related in: MedlinePlus