ROCK-ER activation promotes S-phase entry and increased cell number. (A) Serum-starved, ROCK-ER-expressing NIH 3T3 cells were either left untreated (Control) or treated with 1 μM 4-HT for 18 h either in the presence or in the absence of ROCK inhibitor Y-27632 (10 μM). Cells were pulsed with 10 μM BrdU for 2 h prior to harvest and then collected by trypsinization. Cells were fixed, stained with anti-BrdU antibody and propidium iodide, and then analyzed by flow cytometry. Top panels show BrdU staining versus propidium iodide staining to quantify S-phase cells, and bottom panels show cell numbers versus propidium iodide staining to quantify DNA content. (B) Serum-starved, ROCK-ER-expressing NIH 3T3 cells were either left untreated (Control) or treated with 1 μM 4-HT for 18 h, either in the presence or in the absence of Y-27632 (10 μM) or U0126 (10 μM). Cells were pulsed with 10 μM BrdU for 2 h prior to harvest and then collected by trypsinization. Cells were fixed and stained with anti-BrdU antibody and propidium iodide, flow cytometry was performed, and the cell cycle distribution was analyzed. Mean values ± the standard error of the mean from three repetitions are shown, except for U0126 treatment groups, where mean values from duplicate determinations are reported. Statistical significance was determined by Student's t test, and an asterisk indicates significant difference (P < 0.05) from serum-starved condition. (C) Three independent pools of ROCK-ER-expressing NIH 3T3 cells were serum starved for 24 h and then either left untreated or treated with 1 μM 4-HT for 16 h. The numbers of cells after initial serum starvation and after the additional 16 h without or with 4-HT were determined. Mean values ± the standard error of the mean, normalized to serum-starved cells, are indicated. Statistical significance was determined by Student's t test, and an asterisk indicates significant difference (P < 0.05) from the initial serum-starved condition while a double asterisk indicates significant difference (P < 0.05) from both serum-starved conditions. Results of a representative experiment of two determinations with similar results are shown.

ROCK-ER induction of cyclin D1 by actin-mediated Ras activation. (A) Serum-starved, ROCK-ER-expressing NIH 3T3 cells were left untreated or treated with 1 μM 4-HT for 16 h, either in the presence or in the absence of 1 μM CCD, 0.05 μM SWA, 0.5 μM LTB, or 0.5 μM Jasp. Whole-cell lysates were analyzed by Western blotting for cyclin A, cyclin D1, cyclin E, and phospho-LIMK1/2. Blotting with ERK2 antibody was used to confirm equal protein loading. Results of a representative experiment of three determinations with similar results are shown. (B) Serum-starved, ROCK-ER-expressing NIH 3T3 cells were either left untreated or treated with 1 μM 4-HT or 5% serum as indicated, in the presence or absence of 10 μM Y-27632 or 1 μM CCD, for 16 h. Active Ras-GTP was purified with glutathione S-transferase-Raf-1 RBD agarose beads. GTP-loaded Ras and total Ras were detected by Western blotting. Quantitation is averaged results from three separate experiments; average values ± the standard error of the mean are shown. An asterisk indicates a significant difference, as determined by Student's t test, at P < 0.05. (C) Serum-starved, ROCK-ER-expressing NIH 3T3 cells were either left untreated (Control) or treated with 1 μM 4-HT for 18 h either in the presence or in the absence of 1 μM CCD or 0.5 μM Jasp. Cells were pulsed with 10 μM BrdU for 2 h prior to harvest and then collected by trypsinization. Cells were fixed and stained with anti-BrdU antibody and propidium iodide, flow cytometry was performed, and the cell cycle distribution was analyzed. Mean values ± the standard error of the mean from three repetitions are shown, except for the serum treatment group, where mean values from duplicate determinations are reported. Statistical significance was determined by Student's t test, and an asterisk indicates significant difference (P < 0.05) from the 4-HT-treated condition. (D) Serum-starved, ROCK-ER-expressing NIH 3T3 cells were either left untreated or treated with 1 μM 4-HT for 16 h, either in the presence or in the absence of 10 μM Y-27632 or 10 μM U0126. Cell lysates were immunoblotted for phospho-LIMK1/2, cyclin A, cyclin D1, cyclin E, p27, p21, and phospho-ERK. Equal protein loading was confirmed by blotting with ERK2 antibody. Results of a representative experiment of three determinations with similar results are shown.

Independent effects of ROCK-ER on cyclin A, cyclin D1, and p27 levels. ROCK-ER-expressing NIH 3T3 cells were transfected with control nontargeting (Con) siRNA or siRNA duplexes targeting Lamin A/C, Cyclin A (A), Cyclin D1 (B), or p27 (C). At 24 h posttransfection, cells were trypsinized and replated on plastic tissue culture dishes. Subconfluent cells were serum starved and then either left untreated or treated with 1 μM 4-HT for 16 h. Whole-cell lysates were immunoblotted for phospho-LIMK, cyclin A, cyclin D1, p27, and lamin A/C. Equal protein loading was confirmed by blotting with ERK2 antibody. Results of representative experiments of three or four determinations with similar results are shown.

LIMK2 knockdown abrogates ROCK-ER-induced cyclin A upregulation. (A) ROCK-ER-expressing NIH 3T3 cells were transfected with control nontargeting (Con) siRNA or siRNA(s) against LIMK1, LIMK2, LIMK1 and -2 in combination, or Lamin A/C. At 24 h posttransfection, cells were trypsinized and replated on plastic tissue culture dishes. Subconfluent cells were serum starved and then either left untreated or treated with 1 μM 4-HT for 16 h. Whole-cell lysates were immunoblotted for phospho-LIMK, LIMK 1, LIMK 2, cyclin A, cyclin D1, p27, and lamin A/C. Blotting for ERK2 confirmed equal protein loading. Results of a representative experiment of three determinations with similar results are shown. (B) Model of ROCK regulation of cell cycle proteins. ROCK-ER stimulation leads to activation of Ras and MAPK, which, along with an additional independent pathway, leads to elevation of cyclin D1 expression. Cyclin A is independently elevated in response to ROCK activation via LIMK2. Reduced p27 protein levels were observed following ROCK activation, independent of the effect on cyclin D1, cyclin A, or cell cycle progression.

FIG.1.ROCK-ER activation induces phosphorylation of ROCK substrates and stress fiber formation. (A) Schematic representation of ROCK II showing functional domains (PH = pleckstrin homology domain, CRD = cysteine-rich domain). Conditionally regulated ROCK-ER was generated by fusing amino acids 5 to 553 of ROCK II between the enhanced green fluorescent protein (EGFP) and the estrogen receptor hormone binding domain (hbER). KD-ER was created by changing lysine 121 to glycine [ROCK (K121G)]. (B) NIH 3T3 cells were transduced with retrovirus encoding the conditionally active ROCK-ER fusion proteins. Serum-starved parental NIH 3T3 or ROCK-ER-expressing cells were left untreated or treated with 1 μM 4-HT for 16 h as indicated, either in the presence or in the absence of ROCK inhibitor Y-27632 (10 μM). ROCK II and ROCK-ER protein levels were determined by Western blotting with an antibody against a common epitope. Fusion protein expression was confirmed by blotting with ERα antibody. Results of a representative experiment of three determinations with similar results are shown. (C) NIH 3T3 cells were transduced with retrovirus encoding the conditionally active ROCK-ER or kinase-dead KD-ER fusion protein. Serum-starved parental NIH 3T3, KD-ER-expressing, or ROCK-ER-expressing cells were either left untreated or treated with 1 μM 4-HT for 16 h as indicated, either in the presence or in the absence of ROCK inhibitor Y-27632 (10 μM). Cells were lysed, and protein phosphorylation status was determined with antibodies against phospho-MLC2 (Ser19) and phospho-LIMK1/LIMK2 (antibody recognizing a common epitope at Thr508 and Thr505, respectively). Fusion protein expression was confirmed by blotting with ERα antibody. Results of a representative experiment of three determinations with similar results are shown. (D) Serum-starved, ROCK-ER-expressing NIH 3T3 cells were either left untreated or treated with 1 μM 4-HT for 16 h and then fixed and stained for F-actin (green) and phospho-Ser19 MLC (red). Results of a representative experiment of two determinations with similar results are shown.

Expression of cell cycle regulators following ROCK-ER activation. (A) Serum-starved, KD-ER- and ROCK-ER-expressing NIH 3T3 cells were either left untreated or treated with 1 μM 4-HT for 16 h, either in the presence or in the absence of 10 μM Y-27632. Cell lysates were Western blotted for phospho-LIMK1/2, cyclin A, cyclin D1, cyclin E, p21, and p27. Equal protein loading was confirmed by blotting with ERK2 antibody. Results of a representative experiment of three determinations with similar results are shown. (B) Serum-starved, ROCK-ER-expressing NIH 3T3 cells were either left untreated or treated with 1 μM 4-HT, either in the presence or in the absence of 10 μM Y-27632, for 16 h. Coverslips were fixed, permeabilized, and then stained with anti-cyclin D1 and anti-lamin B1 antibodies. Results of a representative experiment of two determinations with similar results are shown. (C) Serum-starved, ROCK-ER-expressing NIH 3T3 cells were treated with 1 μM 4-HT for the times indicated. Lysates were analyzed by immunoblotting with antibodies against cyclin D1, phospho-LIMK1/2, and ERK2. Results of a representative experiment of three determinations with similar results are shown. EtOH, ethanol.

Adhesion and integrin signaling are not required for ROCK-induced cyclin D1 expression. (A) ROCK-ER-expressing NIH 3T3 cells were plated on uncoated tissue culture dishes or dishes coated with either fibronectin or PDL. Subconfluent cells were serum starved and then left untreated or treated with 1 μM 4-HT for 16 h, either in the presence or in the absence of 10 μM Y-27632 or 10 μM U0126. Cell lysates were Western blotted for phospho-LIMK1/2, cyclin D1, phospho-ERK, ERK2, phospho-MEK1/2 (Ser217/221), MEK1/2, phospho-FAK (Tyr397), and FAK. Results of a representative experiment of three determinations with similar results are shown. (B) ROCK-ER-expressing NIH 3T3 cells were plated on uncoated glass coverslips or PDL-coated coverslips. Subconfluent cells were serum starved overnight and then left in serum-free medium for an additional 16 h. After fixation and permeabilization, cells were stained with anti-paxillin antibody to visualize focal adhesions and with phalloidin to visualize F-actin structures. The scale bar represents 35 μm. Results of a representative experiment of four determinations with similar results are shown. (C) Serum-starved, ROCK-ER-expressing NIH 3T3 cells were trypsinized, resuspended in serum-free DMEM, and replated on either uncoated tissue culture dishes (adherent [Adh]) or poly-HEMA-treated dishes (suspension [Susp]). Cells were left untreated or treated with 1 μM 4-HT for 16 h. Cell lysates were Western blotted for phospho-LIMK1/2, cyclin A, cyclin D1, cyclin E, phospho-ERK, and ERK2. Results of a representative experiment of three determinations with similar results are shown.

Cyclin D1 knockdown inhibits ROCK-ER-induced p107 phosphorylation and cyclin E-associated CDK2 activity. (A) ROCK-ER-expressing NIH 3T3 cells were transfected with control nontargeting (Con) siRNA or siRNA duplexes targeting Cyclin D1. At 24 h posttransfection, cells were trypsinized and replated on plastic tissue culture dishes. Subconfluent cells were serum starved and then either left untreated or treated with 1 μM 4-HT for 16 h. Whole-cell lysates were immunoblotted for phospho-LIMK, p107, and cyclin D1. Equal protein loading was confirmed by blotting with an antibody against ERK2. Results of a representative experiment of two determinations with similar results are shown. (B) Serum-starved, ROCK-ER-expressing NIH 3T3 cells were either left untreated or treated with 1 μM 4-HT for 16 h, either in the presence or in the absence of 10 μM U0126. Cell lysates were immunoblotted for phospho-LIMK, p107, cyclin D1, and phospho-ERK. Equal protein loading was verified by blotting with an antibody against ERK2. Results of a representative experiment of two determinations with similar results are shown. (C) ROCK-ER-expressing NIH 3T3 cells were transfected with control nontargeting (Con) siRNA or siRNA duplexes targeting Cyclin D1. At 24 h posttransfection, cells were trypsinized and replated on plastic tissue culture dishes. Subconfluent cells were serum starved and then either left untreated or treated with 1 μM 4-HT for 16 h. Whole-cell lysates (WCL, lower half) were immunoblotted for phospho-LIMK, cyclin D1, cyclin E, and CDK2. Equal protein loading was confirmed by blotting with ERK2 antibody. Cyclin E was immunoprecipitated (IP), and associated CDK activity was assayed in vitro with histone H1 as the substrate. Cyclin E-associated CDK2 and p21 were revealed by Western blotting (WB) the immunoprecipitated complexes that had been assayed for kinase activity. Results of a representative experiment of two determinations with similar results are shown.