An inherent role of microtubule network in the action of nuclear receptor

Abstract

Estrogen receptor alpha (ERalpha) functions as both a transcription factor and a mediator of rapid estrogen signaling. Recent studies have shown a role for ERalpha-interacting membranous and cytosolic proteins in ERalpha action, but our understanding of the role of the microtubule network in the modulation of ERalpha signaling remains unclear. Here we found that endogenous ERalpha associates with microtubules through the microtubule-binding protein hematopoietic PBX-interaction protein (HPIP). Biochemical and RNA-interference studies demonstrated that HPIP influences ERalpha-dependent rapid estrogen signaling by acting as a scaffold protein and recruits Src kinase and the p85 subunit of phosphatidylinositol 3-kinase to a complex with ERalpha, which in turn stimulates AKT and MAPK. We also found that ERalpha interacts with beta-tubulin through HPIP. Destabilization of microtubules activated ERalpha signaling, whereas stabilization of microtubules repressed ERalpha transcriptional activity in a HPIP-dependent manner. These findings revealed a role for HPIP-microtubule complex in regulating 17beta-estradiol-ERalpha responses in mammalian cells and discovered an inherent role of microtubules in the action of nuclear receptor.

Fig. 1.

ERα interacts with HPIP. (A) A yeast survival assay shows the growth of yeast cells on nutrient selection medium, either in −LT or −AHLT, transformed with ERα and HPIP plasmids. −LT, lack of leucine and tryptophan; −AHLT, lack of adenosine, histidine, leucine, and tryptophan. (B and C) Interaction between T7-ERα and Flag-HPIP in HeLa cells treated with E2 (10 nM) for 5 min. IP, immunoprecipitation. (D and E) Interaction between HPIP and ERα in T7-HPIP-overexpressing MCF-7 cells treated with E2 (10 nM) for 5 min. (F and G) GST pull-down assays show the minimal interacting regions between HPIP and ERα. AF1, activation function 1 domain; DBD, DNA-binding domain; H, hinge region; AF2, activation function 2 domain. (H) Interaction of wild-type HPIP (WT-HPIP) but not mutant HPIP (MT-HPIP) with ERα in MCF-7 cells.

Fig. 2.

HPIP is required for E2-induced ERα-mediated activation of AKT and MAPK. (A) Effect of ICI182,780 on the activation of AKT and MAPK in either pcDNA- or WT-HPIP-transfected MCF-7 cells upon E2 treatment for 15 min. Numbers beneath each lane indicate fold increase in the activation of either AKT or MAPK relative to control. (B) Activation of AKT and MAPK in MCF-7 cells transfected with pcDNA, WT-HPIP, or MT-HPIP in response to E2. (C) Effect of HPIP-specific siRNA upon E2 (10 nM, 15 min) effect on signaling proteins. Con siRNA, control siRNA.

Fig. 3.

HPIP recruits Src and p85 to E2–ERα complex in response to short-term exposure to E2. (A) Effect of E2 (10 nM, 5 min) on the interaction among HPIP, Src, p85 of PI3K, and ERα in HPIP-expressing MCF-7 cells. Cells were treated with ICI182,780 (10⁻⁸ M) for 3 h before E2 treatment. DL, direct lysate. (B) Lysates of MCF-7 cells transfected with either control or HPIP siRNA treated with E2 for 5 min were immunoprecipitated with anti-ERα antibody and blotted with the indicated antibodies. (C and D) Effect of either Src kinase inhibitor PP2 (C) or PI3K inhibitor wortmannin (D) on the activation of AKT and MAPK in MCF-7 cells overexpressing either pcDNA or WT HPIP in response to E2.

Fig. 4.

HPIP tethers ERα to microtubules. (A) Colocalization of T7-HPIP with β-tubulin in MCF-7 cells. (Upper) T7-HPIP (red, labeled with rhodamine), β-tubulin (green, labeled with FITC), and Topro-3-stained DNA (blue). Indicated with white boxes are blow-ups that are shown in Lower. (B) Interaction of HPIP with β-tubulin and ERα. (C) Interaction of ERα with β-tubulin in HepG2 cells treated with E2 (10 nM, 10 min) and/or nocodazole (Noc: 4 μM, 1 h) or Taxol (1 μM, 1 h). (D) HepG2 cells transfected with control or HPIP siRNA and treated with E2 (10 nM, 15 min) were immunoprecipitated with β-tubulin antibody and Western blotted. (E) Nuclear translocation of ERα in either HPIP siRNA-transfected or nocodazole- (4 μM, 4 h) treated HepG2 cells. (F and G) Lysates from MCF-7 cells expressing pcDNA or HPIP treated with colchicine (col; 0.1 μM) (F) or Taxol (0.1 μM) (G) for 1 h before exposure to E2 (10 nM, 15 min) were blotted with the indicated antibodies.

Fig. 5.

HPIP and microtubules negatively regulate ERα transcriptional activity. (A) Effect of T7-HPIP on ERE-luc in MCF-7 cells. (B and C) Effect of nocodazole (Noc; 4 μM, 18 h) or Taxol (1 μM, 18 h) on ERE-luc activity in MCF-7 cells (B) or in HepG2 cells (C). MCF7 cells were treated with Taxol for an additional 1 h in the presence of nocodazole and/or E2 (lanes 7 and 8). (D) Effect of control or HPIP siRNA on ERE-luc activity in HepG2 cells.

Fig. 6.

HPIP promotes breast cancer cell motility and tumorigenesis. (A and B) Uncoated Boyden-chamber assay (A) cell-migration assays (B) were performed with HPIP- and pcDNA-MCF-7 cells treated with E2 (10 nM, 2 days). n = 3. Con, untreated control; ICI, ICI182,780; WRT, wortmannin. (C) Anchorage-independent growth potential of pcDNA or T7-HPIP MCF-7 cells. (D) Induction of tumors in nude mice by MCF-7 cells expressing either pcDNA or HPIP at 8 weeks. (E) A simple model representing the regulation of cytoplasmic and nuclear functions of ERα by HPIP and microtubule (MT) complex.