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The RhoA-Rok-myosin II pathway is involved in extracellular matrix-mediated regulation of prolactin signaling in mammary epithelial cells.

Du JY, Chen MC, Hsu TC, Wang JH, Brackenbury L, Lin TH, Wu YY, Yang Z, Streuli CH, Lee YJ - J. Cell. Physiol. (2012)

Bottom Line: Under these culture conditions, the RhoA pathway is activated, leading to downregulation of prolactin receptor expression and reduced prolactin signaling.In addition, inhibition of myosin II ATPase activity by blebbistatin also exerts a beneficial effect on prolactin receptor expression and prolactin signaling, suggesting that tension exerted by the collagen substratum, in collaboration with the RhoA-Rok-myosin II pathway, contributes to the failure of prolactin signaling.They display high levels of RhoA activity and are inefficient in prolactin signaling, stressing the importance of matrix stiffness in signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology and Immunology, Chung Shan Medical University, Taichung, Taiwan, ROC.

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Related in: MedlinePlus

Application of Y27632 into MECs cultured on collagen I increases Rac activity, whereas expression of constitutively active RhoA in MECs cultured on BM decreases Rac activity. A: MECs cultured on collagen I (CI) were treated with 15 µM Y27632 for 12 h. B: MECs cultured on BM were trypsinized, mock-infected, or infected with adenovirus carrying constitutively active RhoA (Ad-L63RhoA), and replated on BM for 24 h. Cell lysates were incubated with GST-PAK1 p21-binding domain bound to glutathione-agarose beads to precipitate GTP-bound Rac. Total lysates and precipitates were then analyzed by immunoblotting using antibody to Rac. Immunoblots from three independent experiments were analyzed by densitometry. Relative Rac activity is indicated by the amount of Rac-GTP normalized to that of total Rac, and values are expressed as fold stimulation with respect to untreated cells or mock-infected cells. *P < 0.05; **P < 0.01.
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fig07: Application of Y27632 into MECs cultured on collagen I increases Rac activity, whereas expression of constitutively active RhoA in MECs cultured on BM decreases Rac activity. A: MECs cultured on collagen I (CI) were treated with 15 µM Y27632 for 12 h. B: MECs cultured on BM were trypsinized, mock-infected, or infected with adenovirus carrying constitutively active RhoA (Ad-L63RhoA), and replated on BM for 24 h. Cell lysates were incubated with GST-PAK1 p21-binding domain bound to glutathione-agarose beads to precipitate GTP-bound Rac. Total lysates and precipitates were then analyzed by immunoblotting using antibody to Rac. Immunoblots from three independent experiments were analyzed by densitometry. Relative Rac activity is indicated by the amount of Rac-GTP normalized to that of total Rac, and values are expressed as fold stimulation with respect to untreated cells or mock-infected cells. *P < 0.05; **P < 0.01.

Mentions: The mutual antagonism between Rac and Rho GTPases has been well documented (Nimnual et al., 2003; Ohta et al., 2006; Takefuji et al., 2007; Bustos et al., 2008). Given that Rac1 is essential for optimal prolactin signaling in MECs, we asked if the effect of RhoA pathway on prolactin signaling was mediated by inhibiting Rac1 (Akhtar and Streuli, 2006). Rac activity was therefore assessed in response to alterations of RhoA and Rok activity. Inhibition of Rok by Y27632 resulted in elevated Rac activity in cells cultured on collagen I, while constitutively active L63RhoA reduced Rac activity in cells cultured on BM (Fig. 7).


The RhoA-Rok-myosin II pathway is involved in extracellular matrix-mediated regulation of prolactin signaling in mammary epithelial cells.

Du JY, Chen MC, Hsu TC, Wang JH, Brackenbury L, Lin TH, Wu YY, Yang Z, Streuli CH, Lee YJ - J. Cell. Physiol. (2012)

Application of Y27632 into MECs cultured on collagen I increases Rac activity, whereas expression of constitutively active RhoA in MECs cultured on BM decreases Rac activity. A: MECs cultured on collagen I (CI) were treated with 15 µM Y27632 for 12 h. B: MECs cultured on BM were trypsinized, mock-infected, or infected with adenovirus carrying constitutively active RhoA (Ad-L63RhoA), and replated on BM for 24 h. Cell lysates were incubated with GST-PAK1 p21-binding domain bound to glutathione-agarose beads to precipitate GTP-bound Rac. Total lysates and precipitates were then analyzed by immunoblotting using antibody to Rac. Immunoblots from three independent experiments were analyzed by densitometry. Relative Rac activity is indicated by the amount of Rac-GTP normalized to that of total Rac, and values are expressed as fold stimulation with respect to untreated cells or mock-infected cells. *P < 0.05; **P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675639&req=5

fig07: Application of Y27632 into MECs cultured on collagen I increases Rac activity, whereas expression of constitutively active RhoA in MECs cultured on BM decreases Rac activity. A: MECs cultured on collagen I (CI) were treated with 15 µM Y27632 for 12 h. B: MECs cultured on BM were trypsinized, mock-infected, or infected with adenovirus carrying constitutively active RhoA (Ad-L63RhoA), and replated on BM for 24 h. Cell lysates were incubated with GST-PAK1 p21-binding domain bound to glutathione-agarose beads to precipitate GTP-bound Rac. Total lysates and precipitates were then analyzed by immunoblotting using antibody to Rac. Immunoblots from three independent experiments were analyzed by densitometry. Relative Rac activity is indicated by the amount of Rac-GTP normalized to that of total Rac, and values are expressed as fold stimulation with respect to untreated cells or mock-infected cells. *P < 0.05; **P < 0.01.
Mentions: The mutual antagonism between Rac and Rho GTPases has been well documented (Nimnual et al., 2003; Ohta et al., 2006; Takefuji et al., 2007; Bustos et al., 2008). Given that Rac1 is essential for optimal prolactin signaling in MECs, we asked if the effect of RhoA pathway on prolactin signaling was mediated by inhibiting Rac1 (Akhtar and Streuli, 2006). Rac activity was therefore assessed in response to alterations of RhoA and Rok activity. Inhibition of Rok by Y27632 resulted in elevated Rac activity in cells cultured on collagen I, while constitutively active L63RhoA reduced Rac activity in cells cultured on BM (Fig. 7).

Bottom Line: Under these culture conditions, the RhoA pathway is activated, leading to downregulation of prolactin receptor expression and reduced prolactin signaling.In addition, inhibition of myosin II ATPase activity by blebbistatin also exerts a beneficial effect on prolactin receptor expression and prolactin signaling, suggesting that tension exerted by the collagen substratum, in collaboration with the RhoA-Rok-myosin II pathway, contributes to the failure of prolactin signaling.They display high levels of RhoA activity and are inefficient in prolactin signaling, stressing the importance of matrix stiffness in signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology and Immunology, Chung Shan Medical University, Taichung, Taiwan, ROC.

Show MeSH
Related in: MedlinePlus