Increased Ezrin Expression Research Articles

Ezrin Inhibition Overcomes Acquired Resistance to Vemurafenib in BRAFV600E-Mutated Colon Cancer and Melanoma Cells In Vitro.

Despite the advancements in targeted therapy for BRAFV600E-mutated metastatic colorectal cancer (mCRC), the development of resistance to BRAFV600E inhibition limits the response rate and durability of the treatment. Better understanding of the resistance mechanisms to BRAF inhibitors will facilitate the design of novel pharmacological strategies for BRAF-mutated mCRC. The aim of this study was to identify novel protein candidates involved in acquired resistance to BRAFV600E inhibitor vemurafenib in BRAFV600E-mutated colon cancer cells using an integrated proteomics approach. Bioinformatic analysis of obtained proteomics data indicated actin-cytoskeleton linker protein ezrin as a highly ranked protein significantly associated with vemurafenib resistance whose overexpression in the resistant cells was additionally confirmed at the gene and protein level. Ezrin inhibition by NSC305787 increased anti-proliferative and pro-apoptotic effects of vemurafenib in the resistant cells in an additive manner, which was accompanied by downregulation of CD44 expression and inhibition of AKT/c-Myc activities. We also detected an increased ezrin expression in vemurafenib-resistant melanoma cells harbouring the BRAFV600E mutation. Importantly, ezrin inhibition potentiated anti-proliferative and pro-apoptotic effects of vemurafenib in the resistant melanoma cells in a synergistic manner. Altogether, our study suggests a role of ezrin in acquired resistance to vemurafenib in colon cancer and melanoma cells carrying the BRAFV600E mutation and supports further pre-clinical and clinical studies to explore the benefits of combined BRAF inhibitors and actin-targeting drugs as a potential therapeutic approach for BRAFV600E-mutated cancers.

Bisphosphonate-Functionalized Hydroxyapatite Nanoparticles for the Delivery of the Bromodomain Inhibitor JQ1 in the Treatment of Osteosarcoma.

Osteosarcoma (OS) is one of the most common neoplasia among children, and its survival statistics have been stagnating since the combinatorial anticancer therapy triad was first introduced. Here, we report on the assessment of the effect of hydroxyapatite (HAp) nanoparticles loaded with medronate, the simplest bisphosphonate, as a bone-targeting agent and JQ1, a small-molecule bromodomain inhibitor, as a chemotherapeutic in different 2D and 3D K7M2 OS in vitro models. Both additives decreased the crystallinity of HAp, but the effect was more intense for medronate because of its higher affinity for HAp. As the result of PO43--NH+ binding, JQ1 shielded the surface phosphates of HAp and pushed its surface charge to more positive values, exhibiting the opposite effect from calcium-blocking medronate. In contrast to the faster and more exponential release of JQ1 from monetite, its release from HAp nanoparticles followed a zero-order kinetics, but 98% of the payload was released after 48 h. The apoptotic effect of HAp nanoparticles loaded with JQ1, with medronate and with both JQ1 and medronate, was selective in 2D culture: pronounced against the OS cells and nonexistent against the healthy fibroblasts. While OS cell invasion was significantly inhibited by all of the JQ1-containing HAp formulations, that is, with and without medronate, all of the combinations of the targeting compound, medronate, and the chemotherapeutic, JQ1, delivered using HAp, but not HAp alone, inhibited OS cell migration from the tumor spheroids. JQ1 delivered using HAp had an effect on tumor migration, invasion, and apoptosis even at extremely low, subnanomolar concentrations, at which no effect of JQ1 per se was observed, meaning that this form of delivery could help achieve a multifold increase of this drug's efficacy. More than 80% of OS cells internalized JQ1-loaded HAp nanoparticles after 24 h of coincubation, suggesting that this augmentation of the activity of JQ1 may be due to the intracellular delivery and sustained release of the drug enabled by HAp. In addition to the reduction of the OS cell viability, the reduction of the migration and invasion radii was observed in OS tumor spheroids challenged with even JQ1-free medronate-functionalized HAp nanoparticles, demonstrating a definite anticancer activity of medronate alone when combined with HAp. The effect of medronate-functionalized JQ1-loaded HAp nanoparticles was most noticeable against OS cells differentiated into an osteoblastic lineage, in which case they surpassed in effect pure JQ1 and medronate-free compositions. The activity of JQ1 was mediated through increased Ezrin expression and decreased RUNX2 expression and was MYC and FOSL1 independent, but these patterns of gene expression changed in cells challenged with the nanoparticulate form of delivery, having been accompanied by the upregulation of RUNX2 and downregulation of Ezrin in OS cells treated with medronate-functionalized JQ1-loaded HAp nanoparticles.

Vitamin D receptor knockout mice exhibit elongated intestinal microvilli and increased ezrin expression

In addition to its principle function as a calcium regulator, vitamin D can affect cell and tissue morphology. The intestine is an important target tissue of vitamin D, as it must ensure the efficient transport of nutrients across the epithelium while excluding the passage of harmful molecules and bacteria into the organism. These functions require a highly organized morphology, which may be modified by vitamin D deficiency. To elucidate the role of vitamin D in gut morphology and barrier function, we compared the enterocyte microstructures, gut permeability, and cytoskeletal and cell junction protein expression in vitamin D receptor (VDR) knockout (KO) and wild-type (WT) mice. We found that the duodenal epithelial cells in the VDR-KO mice had longer microvilli (+19%) than those of the WT mice (P < .05). Interestingly, microvilli elongation in the VDR-KO mice was associated with higher messenger RNA and protein expression of ezrin, which is involved in the regulation of microvillus morphogenesis. Intestinal tight junction width and permeability were assessed by measuring the fluorescein isothiocyanate dextran concentrations in plasma; the concentrations were comparable between the 2 groups of mice. We further observed a decrease in the messenger RNA and protein expression of the calcium-transporting tight junction protein claudin-2 in the VDR-KO mice compared with the WT mice (P < .05). In conclusion, the mice lacking VDR had longer enterocyte microvilli, likely as a result of increased ezrin expression. However, the morphology of the tight junctions and the intestinal permeability for large molecules were not affected.

Ezrin-expressing lung adenocarcinoma cells and podoplanin-positive fibroblasts form a malignant microenvironment.

Cancer cells and cancer-associated fibroblasts (CAFs) together create the tumor microenvironment, which affects malignant behavior. Lung adenocarcinomas with CAFs expressing podoplanin (PDPN) are clinically aggressive, but the molecular mechanism underlying this phenomenon has not been established. So we identified the characteristic immunophenotype of lung adenocarcinoma cells coexisting with PDPN-expressing CAFs (PDPN-CAFs) and examined how it relates to an aggressive clinicopathological outcome. We analyzed the clinicopathological characteristics of 119 adenocarcinomas with a uniform size (2-3 cm). The expression levels of ten invasiveness-related proteins which related to cell adhesion and invasiveness, such as Ezrin, were examined in cancer cells from PDPN-CAFs (+) cases and from PDPN-CAFs (-) cases (n = 20 each). To examine the functional importance of the identified protein on the invasion phenotype, we performed wound healing and a Matrigel invasion assay using shRNA-knockdown lung adenocarcinoma cells (PC-9). The PDPN-CAFs (+) cases had significantly higher rates of node metastasis (p < 0.01) and vascular invasion (p < 0.01). The cancer cells from the PDPN-CAFs (+) cases also had a significantly higher staining score for Ezrin (p < 0.01) than those from the PDPN-CAFs (-) cases. The migration and invasion activities of the shEzrin-induced PC-9 cells were significantly lower than those of the control cells. Our results indicated that within a tumor microenvironment composed of PDPN-CAFs, increased Ezrin expression in cancer cells might play a key role in the invasiveness of lung adenocarcinoma.

Ezrin expression and cell survival regulation in colorectal cancer

Colorectal cancer (CRC) is the second largest cause of cancer deaths in the United States. A key barrier that prevents better outcomes for this type of cancer as well as other solid tumors is the lack of effective therapies against the metastatic disease. Thus there is an urgent need to fill this gap in cancer therapy. We utilized a 2D-DIGE proteomics approach to identify and characterize proteins that are differentially regulated between primary colon tumor and liver metastatic deposits of the IGF1R-dependent GEO human CRC xenograft, orthotopically implanted in athymic nude mice that may serve as potential therapeutic targets against CRC metastasis. We observed increased expression of ezrin in liver metastasis in comparison to the primary colonic tumor. Increased ezrin expression was further confirmed by western blot and microarray analyses. Ezrin, a cytoskeletal protein belonging to Ezrin–Radixin–Moesin (ERM) family plays important roles in cell motility, invasion and metastasis. However, its exact function in colorectal cancer is not well characterized. Establishment of advanced GEO cell lines with enhanced liver-metastasizing ability showed a significant increase in ezrin expression in liver metastasis. Increased phosphorylation of ezrin at the T567 site (termed here as p-ezrin T567) was observed in liver metastasis. IHC studies of human CRC patient specimens showed an increased expression of p-ezrin T567 in liver metastasis compared to the primary tumors of the same patient. Ezrin modulation by siRNA, inhibitors and T567A/D point mutations significantly downregulated inhibitors of apoptosis (IAP) proteins XIAP and survivin that have been linked to increased aberrant cell survival and metastasis and increased cell death. Inhibition of the IGF1R signaling pathway by humanized recombinant IGF1R monoclonal antibody MK-0646 in athymic mouse subcutaneous xenografts resulted in inhibition of p-ezrin T567 indicating ezrin signaling is downstream of the IGF1R signaling pathway. We identified increased expression of p-ezrin T567 in CRC liver metastasis in both orthotopically implanted GEO tumors as well as human patient specimens. We report for the first time that p-ezrin T567 is downstream of the IGF1R signaling and demonstrate that ezrin regulates cell survival through survivin/XIAP modulation.

Ezrin Overexpression by Transformed Human Ovarian Surface Epithelial Cells, Ovarian Cleft Cells, and Serous Ovarian Adenocarcinoma Cells

We have shown that ezrin expression correlates with ovarian epithelial cancer (OVCA) cell proliferation and metastatic behavior. In this study, we evaluated ezrin expression in transformed ovarian superficial epithelial cells (OSE) in ovarian clefts and in culture. Immunohistochemistry and Western blotting for immunoreactive ezrin (ir-ezrin) in normal ovarian tissue, cultured OSE, and ovarian epithelial cancer cells. While ir-ezrin was not demonstrable in normal cuboidal surface cells or interior ovarian organelles, cells lining the ovarian clefts strongly expressed ir-ezrin. Long-term culture of OSE increased ezrin expression and cytological abnormalities. Administration of estradiol and insulin at levels reported in inclusions dramatically induced OSE ir-ezrin expression to OVCA levels and membrane specializations; ruffling, pseudopodia and filopodia. Moreover epidermal growth factor (EGF) drastically increased ezrin translocation in OSE cells in a time-dependent manner. Ezrin expression by OSE increases during transformation. Ezrin expression is responsive to estradiol and growth factors previously shown to be present in ovarian inclusions. These findings suggest that the microenvironment in ovarian inclusions and clefts contributes to the development of OVCA. Our findings elaborate on the mechanism of the ovarian origin of OVCA.

Potential transcriptional regulatory regions exist upstream of the human ezrin gene promoter in esophageal carcinoma cells

We previously demonstrated that the region -87/+134 of the human ezrin gene (VIL2) exhibited promoter activity in human esophageal carcinoma EC109 cells, and a further upstream region -1324/-890 positively regulated transcription. In this study, to identify the transcriptional regulatory regions upstream of the VIL2 promoter, we cloned VIL2 -1541/-706 segment containing the -1324/-890, and investigated its transcriptional regulatory properties via luciferase assays in transiently transfected cells. In EC109 cells, it was found that VIL2 -1541/-706 possessed promoter and enhancer activities. We also localized transcriptional regulatory regions by fusing 5'- or 3'-deletion segments of VIL2 -1541/-706 to a luciferase reporter. We found that there were three positive and one negative transcriptional regulatory regions within VIL2 -1541/-706 in EC109 cells. When these regions were separately located upstream of the luciferase gene without promoter, or located upstream of the VIL2 promoter or SV40 promoter directing the luciferase gene, only VIL2 -1297/-1186 exhibited considerable promoter and enhancer activities, which were lower than those of -1541/-706. In addition, transient expression of Sp1 increased ezrin expression and the transcriptional activation of VIL2 -1297/-1186. Other three regions, although exhibiting significantly positive or negative transcriptional regulation in deletion experiments, showed a weaker or absent regulation. These data suggested that more than one region upstream of the VIL2 promoter participated in VIL2 transcription, and the VIL2 -1297/-1186, probably as a key transcriptional regulatory region, regulated VIL2 transcription in company with other potential regulatory regions.

Expression of ezrin correlates with poor prognosis of nasopharyngeal carcinoma

A significant proportion of patients with nasopharyngeal carcinoma (NPC) will develop regional relapse or distant metastasis after treatment. So, to find valuable prognostic factors becomes very important. To elucidate the expression of ezrin in NPC and its correlation with patients' clinicopathologic characteristics, time to progression, and overall survival, immunohistochemistry stains for ezrin were applied to 200 tissue specimens of NPC. Ezrin expression had no significant correlation with sex, age, primary tumor stage (T), Epstein-Barr virus (EBV) VCA-IgA antibody, EBV EA-IgA antibody, and EBV DNA copy except primary nodal status (N; p= .032). Increased ezrin expression was significantly related to higher lymph nodal metastatic rate (p< .004). Among ezrin (negative/weak), ezrin (moderate), and ezrin (intense) groups, there was a significant difference for median time to progression (not reached vs. 63.2 months vs. 46 months, P< .001) and median overall survival (100.7 months vs. 80.8 months vs. 70.6 months, P< .001). Ezrin expression status was an independent prognostic factor in multivariate (Cox) analysis. Ezrin, which was significantly correlated to prognosis of disease, may be involved in the tumorigenesis, progression, and invasion of NPC. Thus, our data offer the possibility of future therapeutic targets.

Sp1 and AP-1 Regulate Expression of the Human Gene VIL2 in Esophageal Carcinoma Cells

Ezrin, encoded by VIL2, is a membrane-cytoskeletal linker protein that has been suggested to be involved in tumorigenesis. Ezrin expression in esophageal squamous cell carcinoma (ESCC) was described recently, but its clinical significance and the molecular mechanism underlying its regulated expression remain unclear. Thus, we retrospectively evaluated ezrin expression by immunohistochemistry in a tissue microarray representing 193 ESCCs. Ezrin overexpression in 90 of 193 tumors (46.6%) was associated with poor survival (p = 0.048). We then explored the mechanism by which ezrin expression is controlled in ESCC by assessing the transcriptional regulatory regions of human VIL2 by fusing deletions or site-directed mutants of the 5'-flanking region of the gene to a luciferase reporter. We found that the region -87/-32 containing consensus Sp1 (-75/-69) and AP-1 (-64/-58) binding sites is crucial for VIL2 promoter activity in esophageal carcinoma cells (EC109) derived from ESCC. AP-1 is comprised of c-Jun and c-Fos. Electrophoretic mobility shift and chromatin immunoprecipitation experiments demonstrated that Sp1 and c-Jun bound specifically to their respective binding sites within the VIL2 promoter. In addition, transient expression of Sp1, c-Jun, or c-Fos increased ezrin expression and VIL2 promoter activity. Use of selective inhibitors revealed that VIL2 transactivation required the MEK1/2 signal transduction pathway but not JNK or p38 MAPK. Taken together, we propose a possible signal transduction pathway whereby MEK1/2 phosphorylates ERK1/2, which phosphorylates Sp1 and AP-1 that in turn bind to their respective binding sites to regulate the expression of human VIL2 in ESCC cells.

Intense cytoplasmic ezrin immunoreactivity predicts poor survival in colorectal cancer

Ezrin is a membrane-cytoskeleton anchor, which, in experimental models, regulates tumor cell invasion and metastatic ability. We carried out immunohistochemical analysis of ezrin in 74 advanced colorectal cancer patients and correlated it to clinicopathologic variables and disease outcome. In contrast to the predominantly membraneous immunoreactivity of normal colorectal epithelium, ezrin expression in the colorectal cells was typically cytoplasmic. Altogether, 16.2% (12/74) of the tumors showed negative/weak ezrin staining, 35.1% (26/74) had moderate staining, and 48.6% (36/74) had intense staining. The expression was more intense in colon than in rectal carcinomas (P = .003). Increased ezrin expression was associated with adverse outcome, that is, shorter disease-specific survival; 48.3 months and 36.6 months for negative-weak versus intense expression (P = .041) as well as shorter survival with metastases at 36 months (P = .030); the metastases(36) rates in ezrin(neg/weak), ezrin(moderate), ezrin(intense) are 58.3%, 25.0%, and 18.4%, respectively. In univariate survival analysis, dichotomized (negative/weak versus moderate/strong) ezrin expression significantly predicted both the 5-year disease specific survival (P = .035) and 5-year metastases (P = .018) but lost this predictive power in multivariate (Cox) analysis. High ezrin expression was also related to high E-cadherin (cytoplasmic) expression, DNA aneuploidy, and high thymidylate synthase expression (P = .046, P = .042, P = .046, respectively). These results suggest that ezrin may play a role in colorectal cancer progression and that ezrin expression might provide clinically valuable information in predicting the biological behavior of colorectal cancer.

Starvation Induces Phase-Specific Changes in the Proteome of Mouse Small Intestine

Food deprivation results in metabolic, structural, and functional changes in the small intestine that influences gut mucosal integrity, epithelial cell proliferation, mucin synthesis, and other processes. The underlying mechanisms are still unclear, which lead to the study of molecular effects of short-term and long-term starvation in the intestine of mice. A comparative proteomics approach, combining two-dimensional gel electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, was used to identify intestinal proteins whose expression is changed under different starvation conditions (0, 12, 24, and 72 h). In total, the expression levels of 80 protein spots changed significantly between the different groups. The results demonstrate that after 12 h of starvation, mainly proteins involved in glycolysis and energy metabolism show decreased expression levels. Starvation for 24 h results in a down-regulation of proteins involved in protein synthesis and amino acid metabolism. Simultaneously, proteins with a protective role, e.g., reg I and II, glutathione peroxidase 3, and carbonic anhydrase 3, are clearly up-regulated. The last starvation phase (72 h) is characterized by increased ezrin expression, which may enhance villus morphogenesis critical for survival. Together, these results provide novel insights in the intestinal starvation response and may contribute to improved nutritional support during conditions characterized by malnutrition.

Altered expression of selected genes in kidney of rats with lithium-induced NDI

Lithium treatment is associated with development of nephrogenic diabetes insipidus, caused in part by downregulation of collecting duct aquaporin-2 (AQP2) and AQP3 expression. In the present study, we carried out cDNA microarray screening of gene expression in the inner medulla (IM) of lithium-treated and control rats, and selected genes were then investigated at the protein level by immunoblotting and/or immunohistochemistry. The following genes exhibited significantly altered transcription and mRNA expression levels, and these were compatible with the changes in protein expression. 11beta-Hydroxysteroid dehydrogenase type 2 protein expression in the IM was markedly increased (198 +/- 25% of controls, n = 6), and immunocytochemistry demonstrated an increased labeling of IM collecting duct (IMCD) principal cells. This indicated altered renal mineralocorticoid/glucocorticoid responses in lithium-treated rats. The inhibitor of cyclin-dependent kinases p27 (KIP) protein expression was significantly decreased or undetectable in the IMCD cells, pointing to increased cellular proliferation and remodeling. Heat shock protein 27 protein expression was decreased in the IM (64 +/- 6% of controls, n = 6), likely to be associated with the decreased medullary osmolality in lithium-treated rats. Consistent with this, lens aldose reductase protein expression was markedly decreased in the IM (16 +/- 2% of controls, n = 6), and immunocytochemistry revealed decreased expression in the thin limb cells in the middle and terminal parts of the IM. Ezrin protein expression was upregulated in the IM (158 +/- 16% of controls, n = 6), where it was predominantly expressed in the apical and cytoplasmic domain of the IMCD cells. Increased ezrin expression indicated remodeling of the actin cytoskeleton and/or altered regulation of IMCD transporters. In conclusion, the present study demonstrates changes in gene expression not only in the collecting duct but also in the thin limb of the loop of Henle in the IM, and several of these genes are linked to altered sodium and water reabsorption, cell cycling, and changes in interstitial osmolality.

Ezrin expression in tissue microarray of primary and recurrent gliomas.

Malignant progression, infiltrative growth pattern and recurrencies after surgery are characteristic features of diffuse gliomas. Ezrin is a membrane-cytoskeleton linker protein expressed in several types of neoplasms. In experimental models, increased ezrin expression correlates with invasion of malignant glioma cells. We studied ezrin expression and its correlation with patient survival in 229 primary and recurrent astrocytomas (WHO grades II-IV), oligodendrogliomas (II-III) and oligoastrocytomas (II-III) of 113 patients. Ezrin expression as evaluated by immunohistochemistry and immunoblotting was detected in all studied glioma types. Staining intensity and number of immunoreactive cells correlated with increasing malignancy of astrocytomas and oligoastrocytomas (P = 0.001). Ezrin expression was strongest in astrocytomas (P = 0.006). Also oligodendrogliomas were positive for ezrin. High ezrin expression in primary gliomas correlated with shorter time to recurrence (P < 0.05) and poor overall survival (P < 0.05) of the patients. Ezrin expression increased during progression of the tumours (P < 0.05). However, ezrin was not an independent prognostic factor. The results of this study show that ezrin expression is associated with progression of gliomas and correlates with histological cell type and WHO tumour grade.

Ezrin immunoreactivity in relation to survival in serous ovarian carcinoma patients

Objective Ezrin is a membrane–cytoskeleton linker protein, which regulates cell polarity and signaling. Increased ezrin expression in astrocytomas and uveal melanomas is correlated with unfavorable prognostic factors and reduced patient survival. We investigated ezrin IR in normal ovarian surface epithelium and serous ovarian carcinomas, and its relation with clinical parameters and patient outcome. Methods Tissue microarray blocks were constructed of all serous ovarian carcinoma tissue samples removed at a primary operation in Helsinki University Central Hospital between 1964 and 1999 and of healthy ovarian tissue samples. Ezrin expression was assessed by indirect immunohistochemistry using a monoclonal 3C12 ezrin antibody. Tissue samples ( n = 440) were scored for the intensity of ezrin immunoreactivity, and the scores were compared with patient age, the stage and grade of disease, and disease outcome. Results Healthy ovarian epithelium showed strong polarized ezrin immunoreactivity. In serous ovarian carcinoma, the reactivity varied from strong (15.0% of samples) to moderate (57.3%) or weak/negative (27.7%) and the subcellular distribution was typically diffuse. Weak or negative expression of ezrin was associated with shorter survival ( P = 0.027) but also with an advanced age of the patients ( P = 0.0001), and a higher histological grade of the disease ( P = 0.032). In Cox multivariate survival analysis, ezrin immunoreactivity had no independent effect on survival, when controlling for the stage and grade of the disease, and patient age. Conclusions In contrast with astrocytomas and uveal melanomas, negative or weak ezrin immunoreactivity in serous ovarian carcinoma correlates with poor patient outcome.

Increased Ezrin Expression and Activation by CDK5 Coincident with Acquisition of the Senescent Phenotype

Passage of normal cells in culture leads to senescence, an irreversible cell cycle exit characterized by biochemical changes and a distinctive morphology. Cellular stresses, including oncogene activation, can also lead to senescence. Consistent with an antioncogenic role for this process, the tumor suppressor pRb plays a critical role in senescence. Reexpression of pRb in human tumor cells results in senescence-like changes including cell cycle exit and shape changes. Here we show that senescence is accompanied by increased expression and altered localization of ezrin, an actin binding protein involved in membrane-cytoskeletal signaling. pRb expression results in the stimulation of CDK5-mediated phosphorylation of ezrin with subsequent membrane association and induction of cell shape changes, linking pRb activity to cytoskeletal regulation in senescent cells.

Tissue-specific regulation by estrogen of ezrin and ezrin/radixin/moesin-binding protein 50.

The morphology and function of rat GH3 pituitary cells are profoundly affected by estradiol-17beta (E2), presumably due to changes in the profile of gene expression. We recently reported that a major target of E2 in these cells is the ezrin gene, which encodes a cytoskeletal linker protein that forms a complex with ezrin/radixin/ moesin-binding protein 50 (EBP50) in some cell types. Other studies have shown that EBP50 levels are increased by E2 in human breast and uterine tissue. Thus, we examined whether ezrin and EBP50 expression is coordinately increased by E2 in GH3 cells in vitro and rat pituitary glands in vivo. Ezrin levels are repressed by the steroidal antiestrogen, ICI 182780, and this effect is abrogated by E2 and the ERalpha-specific agonist, PPT, in GH3 cells. In contrast, EBP50 levels remained constant during these treatments. Ezrin and EBP50 did not display extensive colocalization. Moreover, ezrin was not co-immunoprecipitated by an EBP50 antibody in parental GH3 cells or in GH3 cells stably overexpressing EBP50, but was co-immunoprecipitated with EBP50 in human breast MCF-7 cells. Disruption of the actin cytoskeleton of GH3 cells changed the distribution of ezrin within subcellular fractions, but had no effect on EBP50. Finally, in juvenile female rats, E2 injections increased ezrin expression in the pituitary and uterus, but increased EBP50 expression only in the uterus. These findings demonstrate tissue specificity in the formation of ezrin-EBP50 complexes and in the regulation of EBP50 expression in estrogen-responsive tissues.