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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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Constitutively active RhoA inhibits prolactin signaling in MECs cultured on BM. MECs cultured on BM were trypsinized, infected with adenovirus carrying HA-tagged constitutively active RhoA (Ad-L63RhoA) or LacZ (Ad-LacZ), and replated on BM for 24 h. A: Cells were then stimulated with 3 µg/ml prolactin in the absence or presence 100 µM ZVAD-fmk for 24 h. Total RNA was extracted, reverse transcribed, and PCR-amplified with primers for prolactin receptor, β-casein, and GAPDH. B: Cells were serum-starved for 8 h, and then stimulated with prolactin for 15 min. Cell lysates were analyzed by immunoblotting with antibodies to phospho-Stat5 (p-Stat5) and Stat5. C: Cells were stimulated with prolactin in the absence or presence ZVAD-fmk for 36 h. Total cell lysates were analyzed by immunoblotting with antibodies to β-casein, Erk, and HA. Levels of Erk were used as loading control.
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fig03: Constitutively active RhoA inhibits prolactin signaling in MECs cultured on BM. MECs cultured on BM were trypsinized, infected with adenovirus carrying HA-tagged constitutively active RhoA (Ad-L63RhoA) or LacZ (Ad-LacZ), and replated on BM for 24 h. A: Cells were then stimulated with 3 µg/ml prolactin in the absence or presence 100 µM ZVAD-fmk for 24 h. Total RNA was extracted, reverse transcribed, and PCR-amplified with primers for prolactin receptor, β-casein, and GAPDH. B: Cells were serum-starved for 8 h, and then stimulated with prolactin for 15 min. Cell lysates were analyzed by immunoblotting with antibodies to phospho-Stat5 (p-Stat5) and Stat5. C: Cells were stimulated with prolactin in the absence or presence ZVAD-fmk for 36 h. Total cell lysates were analyzed by immunoblotting with antibodies to β-casein, Erk, and HA. Levels of Erk were used as loading control.

Mentions: Since RhoA inhibits insulin signaling in MECs, we reasoned that it might have a similar effect on prolactin signaling, either at the level of prolactin receptor expression or further downstream. We therefore expressed constitutively active RhoA (L63RhoA) in MECs cultured on BM. This diminished the expression of prolactin receptor, and completely prevented prolactin-induced Stat5 tyrosine phosphorylation and β-casein expression (Fig. 3). To confirm that RhoA-mediated inhibition of prolactin signaling is not secondary to the induction of apoptosis, ZVAD-fmk was added to the cultures to prevent caspase activation. ZVAD-fmk did not eliminate the inhibitory effect of RhoA on β-casein expression (Fig. 3). Thus, RhoA activation prevents prolactin receptor expression and Stat5 signaling.


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)

Constitutively active RhoA inhibits prolactin signaling in MECs cultured on BM. MECs cultured on BM were trypsinized, infected with adenovirus carrying HA-tagged constitutively active RhoA (Ad-L63RhoA) or LacZ (Ad-LacZ), and replated on BM for 24 h. A: Cells were then stimulated with 3 µg/ml prolactin in the absence or presence 100 µM ZVAD-fmk for 24 h. Total RNA was extracted, reverse transcribed, and PCR-amplified with primers for prolactin receptor, β-casein, and GAPDH. B: Cells were serum-starved for 8 h, and then stimulated with prolactin for 15 min. Cell lysates were analyzed by immunoblotting with antibodies to phospho-Stat5 (p-Stat5) and Stat5. C: Cells were stimulated with prolactin in the absence or presence ZVAD-fmk for 36 h. Total cell lysates were analyzed by immunoblotting with antibodies to β-casein, Erk, and HA. Levels of Erk were used as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig03: Constitutively active RhoA inhibits prolactin signaling in MECs cultured on BM. MECs cultured on BM were trypsinized, infected with adenovirus carrying HA-tagged constitutively active RhoA (Ad-L63RhoA) or LacZ (Ad-LacZ), and replated on BM for 24 h. A: Cells were then stimulated with 3 µg/ml prolactin in the absence or presence 100 µM ZVAD-fmk for 24 h. Total RNA was extracted, reverse transcribed, and PCR-amplified with primers for prolactin receptor, β-casein, and GAPDH. B: Cells were serum-starved for 8 h, and then stimulated with prolactin for 15 min. Cell lysates were analyzed by immunoblotting with antibodies to phospho-Stat5 (p-Stat5) and Stat5. C: Cells were stimulated with prolactin in the absence or presence ZVAD-fmk for 36 h. Total cell lysates were analyzed by immunoblotting with antibodies to β-casein, Erk, and HA. Levels of Erk were used as loading control.
Mentions: Since RhoA inhibits insulin signaling in MECs, we reasoned that it might have a similar effect on prolactin signaling, either at the level of prolactin receptor expression or further downstream. We therefore expressed constitutively active RhoA (L63RhoA) in MECs cultured on BM. This diminished the expression of prolactin receptor, and completely prevented prolactin-induced Stat5 tyrosine phosphorylation and β-casein expression (Fig. 3). To confirm that RhoA-mediated inhibition of prolactin signaling is not secondary to the induction of apoptosis, ZVAD-fmk was added to the cultures to prevent caspase activation. ZVAD-fmk did not eliminate the inhibitory effect of RhoA on β-casein expression (Fig. 3). Thus, RhoA activation prevents prolactin receptor expression and Stat5 signaling.

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