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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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MECs cultured on 2D collagen I and 2D laminin are less effective in prolactin signaling than those cultured on 3D BM. MECs were cultured on 2D collagen I (CI), 2D laminin (LN), or 3D BM for 4 days. A: Phase contrast micrographs of the cells. B: Cell lysates were incubated with GST-Rhotekin Rho binding domain bound to glutathione-agarose beads to precipitate GTP-bound Rho. Total lysates and precipitates were then analyzed by immunoblotting using antibody to RhoA. C: Total RNA was reverse transcribed and PCR-amplified with primers for prolactin receptor and GAPDH. D: Total RNA was reverse transcribed and subjected to real-time PCR using primers for prolactin receptor and MAPK1. Relative expression of prolactin receptor normalized to MAPK1 is expressed as fold increase with respect to cells cultured on collagen I (n = 3). E: Cells were serum-starved for 8 h, and then stimulated without or with 3 µg/ml prolactin for 15 min. Cell lysates were analyzed by immunoblotting with antibodies to phospho-Stat5 (p-Stat5) and Stat5. F: Cells were stimulated without or with prolactin for 36 h. Total cell lysates were analyzed by immunoblotting with antibodies to β-casein and Erk.
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fig08: MECs cultured on 2D collagen I and 2D laminin are less effective in prolactin signaling than those cultured on 3D BM. MECs were cultured on 2D collagen I (CI), 2D laminin (LN), or 3D BM for 4 days. A: Phase contrast micrographs of the cells. B: Cell lysates were incubated with GST-Rhotekin Rho binding domain bound to glutathione-agarose beads to precipitate GTP-bound Rho. Total lysates and precipitates were then analyzed by immunoblotting using antibody to RhoA. C: Total RNA was reverse transcribed and PCR-amplified with primers for prolactin receptor and GAPDH. D: Total RNA was reverse transcribed and subjected to real-time PCR using primers for prolactin receptor and MAPK1. Relative expression of prolactin receptor normalized to MAPK1 is expressed as fold increase with respect to cells cultured on collagen I (n = 3). E: Cells were serum-starved for 8 h, and then stimulated without or with 3 µg/ml prolactin for 15 min. Cell lysates were analyzed by immunoblotting with antibodies to phospho-Stat5 (p-Stat5) and Stat5. F: Cells were stimulated without or with prolactin for 36 h. Total cell lysates were analyzed by immunoblotting with antibodies to β-casein and Erk.

Mentions: Our results show that prolactin signaling in MECs cultured on the soft BM hydrogel is more efficient than signaling in cells cultured on the rigid surface of collagen I-coated plastic. These culture conditions differ in both matrix rigidity and composition. Since laminin is the major component of BM and is essential for prolactin signaling, we examined prolactin signaling in MECs cultured on laminin-coated plastic, which has about the same rigidity as collagen I-coated dishes. This would help to show whether matrix stiffness affects prolactin signaling. Similar to MECs cultured on 2D collagen I, cells on 2D laminin formed monolayers, and exhibited higher RhoA activity than those cultured on 3D BM (Fig. 8A,B).


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)

MECs cultured on 2D collagen I and 2D laminin are less effective in prolactin signaling than those cultured on 3D BM. MECs were cultured on 2D collagen I (CI), 2D laminin (LN), or 3D BM for 4 days. A: Phase contrast micrographs of the cells. B: Cell lysates were incubated with GST-Rhotekin Rho binding domain bound to glutathione-agarose beads to precipitate GTP-bound Rho. Total lysates and precipitates were then analyzed by immunoblotting using antibody to RhoA. C: Total RNA was reverse transcribed and PCR-amplified with primers for prolactin receptor and GAPDH. D: Total RNA was reverse transcribed and subjected to real-time PCR using primers for prolactin receptor and MAPK1. Relative expression of prolactin receptor normalized to MAPK1 is expressed as fold increase with respect to cells cultured on collagen I (n = 3). E: Cells were serum-starved for 8 h, and then stimulated without or with 3 µg/ml prolactin for 15 min. Cell lysates were analyzed by immunoblotting with antibodies to phospho-Stat5 (p-Stat5) and Stat5. F: Cells were stimulated without or with prolactin for 36 h. Total cell lysates were analyzed by immunoblotting with antibodies to β-casein and Erk.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig08: MECs cultured on 2D collagen I and 2D laminin are less effective in prolactin signaling than those cultured on 3D BM. MECs were cultured on 2D collagen I (CI), 2D laminin (LN), or 3D BM for 4 days. A: Phase contrast micrographs of the cells. B: Cell lysates were incubated with GST-Rhotekin Rho binding domain bound to glutathione-agarose beads to precipitate GTP-bound Rho. Total lysates and precipitates were then analyzed by immunoblotting using antibody to RhoA. C: Total RNA was reverse transcribed and PCR-amplified with primers for prolactin receptor and GAPDH. D: Total RNA was reverse transcribed and subjected to real-time PCR using primers for prolactin receptor and MAPK1. Relative expression of prolactin receptor normalized to MAPK1 is expressed as fold increase with respect to cells cultured on collagen I (n = 3). E: Cells were serum-starved for 8 h, and then stimulated without or with 3 µg/ml prolactin for 15 min. Cell lysates were analyzed by immunoblotting with antibodies to phospho-Stat5 (p-Stat5) and Stat5. F: Cells were stimulated without or with prolactin for 36 h. Total cell lysates were analyzed by immunoblotting with antibodies to β-casein and Erk.
Mentions: Our results show that prolactin signaling in MECs cultured on the soft BM hydrogel is more efficient than signaling in cells cultured on the rigid surface of collagen I-coated plastic. These culture conditions differ in both matrix rigidity and composition. Since laminin is the major component of BM and is essential for prolactin signaling, we examined prolactin signaling in MECs cultured on laminin-coated plastic, which has about the same rigidity as collagen I-coated dishes. This would help to show whether matrix stiffness affects prolactin signaling. Similar to MECs cultured on 2D collagen I, cells on 2D laminin formed monolayers, and exhibited higher RhoA activity than those cultured on 3D BM (Fig. 8A,B).

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