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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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Expression of dominant negative Rok or application of Rok inhibitor Y27632 enhances prolactin signaling in MECs cultured on collagen I. A–C: MECs cultured on collagen I (CI) were infected with adenovirus carrying HA-tagged dominant negative Rok (Ad-RB/PH (TT)) or LacZ (Ad-LacZ) in situ for 24 h. D–F: MECs cultured on collagen I were pretreated with 10 µM Y27632. A,D: Cells were then stimulated without or with 3 µg/ml prolactin for 24 h. Total RNA was isolated, reverse transcribed, and PCR-amplified with primers for prolactin receptor, β-casein, and GAPDH. B,E: Cells were stimulated without or with prolactin for 15 min. Cell lysates were analyzed by immunoblotting with antibodies to phospho-Stat5 (p-Stat5), Stat5 or HA. C,F: Cells were stimulated without or with prolactin for 36 h. Total cell lysates were analyzed by immunoblotting with antibodies to β-casein, Erk, or HA.
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fig05: Expression of dominant negative Rok or application of Rok inhibitor Y27632 enhances prolactin signaling in MECs cultured on collagen I. A–C: MECs cultured on collagen I (CI) were infected with adenovirus carrying HA-tagged dominant negative Rok (Ad-RB/PH (TT)) or LacZ (Ad-LacZ) in situ for 24 h. D–F: MECs cultured on collagen I were pretreated with 10 µM Y27632. A,D: Cells were then stimulated without or with 3 µg/ml prolactin for 24 h. Total RNA was isolated, reverse transcribed, and PCR-amplified with primers for prolactin receptor, β-casein, and GAPDH. B,E: Cells were stimulated without or with prolactin for 15 min. Cell lysates were analyzed by immunoblotting with antibodies to phospho-Stat5 (p-Stat5), Stat5 or HA. C,F: Cells were stimulated without or with prolactin for 36 h. Total cell lysates were analyzed by immunoblotting with antibodies to β-casein, Erk, or HA.

Mentions: Rok is a key effector of RhoA, and we therefore tested its involvement in the inhibition of prolactin signaling in MECs cultured on collagen I. For these experiments, we either added the Rok inhibitor, Y27632 to cultures, or expressed dominant negative Rok (RB/PH (TT)). In both cases, blocking the action of Rok enhanced prolactin receptor expression (Fig. 5A), as well as prolactin-induced Stat5 phosphorylation (Fig. 5B) and β-casein expression (Fig. 5A,C).


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)

Expression of dominant negative Rok or application of Rok inhibitor Y27632 enhances prolactin signaling in MECs cultured on collagen I. A–C: MECs cultured on collagen I (CI) were infected with adenovirus carrying HA-tagged dominant negative Rok (Ad-RB/PH (TT)) or LacZ (Ad-LacZ) in situ for 24 h. D–F: MECs cultured on collagen I were pretreated with 10 µM Y27632. A,D: Cells were then stimulated without or with 3 µg/ml prolactin for 24 h. Total RNA was isolated, reverse transcribed, and PCR-amplified with primers for prolactin receptor, β-casein, and GAPDH. B,E: Cells were stimulated without or with prolactin for 15 min. Cell lysates were analyzed by immunoblotting with antibodies to phospho-Stat5 (p-Stat5), Stat5 or HA. C,F: Cells were stimulated without or with prolactin for 36 h. Total cell lysates were analyzed by immunoblotting with antibodies to β-casein, Erk, or HA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig05: Expression of dominant negative Rok or application of Rok inhibitor Y27632 enhances prolactin signaling in MECs cultured on collagen I. A–C: MECs cultured on collagen I (CI) were infected with adenovirus carrying HA-tagged dominant negative Rok (Ad-RB/PH (TT)) or LacZ (Ad-LacZ) in situ for 24 h. D–F: MECs cultured on collagen I were pretreated with 10 µM Y27632. A,D: Cells were then stimulated without or with 3 µg/ml prolactin for 24 h. Total RNA was isolated, reverse transcribed, and PCR-amplified with primers for prolactin receptor, β-casein, and GAPDH. B,E: Cells were stimulated without or with prolactin for 15 min. Cell lysates were analyzed by immunoblotting with antibodies to phospho-Stat5 (p-Stat5), Stat5 or HA. C,F: Cells were stimulated without or with prolactin for 36 h. Total cell lysates were analyzed by immunoblotting with antibodies to β-casein, Erk, or HA.
Mentions: Rok is a key effector of RhoA, and we therefore tested its involvement in the inhibition of prolactin signaling in MECs cultured on collagen I. For these experiments, we either added the Rok inhibitor, Y27632 to cultures, or expressed dominant negative Rok (RB/PH (TT)). In both cases, blocking the action of Rok enhanced prolactin receptor expression (Fig. 5A), as well as prolactin-induced Stat5 phosphorylation (Fig. 5B) and β-casein expression (Fig. 5A,C).

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