Campbell Celeste H, Bulayeva Nataliya, Brown David B, Gametchu Bahiru, Watson Cheryl S
Human Biological Chemistry and Genetics Department, University of Texas, Medical Branch, Galveston Texas 77555-0645, USA.
FASEB J. 2002 Dec;16(14):1917-27. doi: 10.1096/fj.02-0182com.
We used modified immunocytochemical conditions to quantify a membrane form of estrogen receptor-alpha (mERalpha) in a rat pituitary tumor cell line, GH3/B6/F10. We studied the regulation of mERalpha vs. levels of intracellular ERalpha (iERalpha) using our 96-well plate immunoassay. The anti-ERalpha antibody C542 was used to label the ERalpha (via conjugated alkaline phosphatase) in fixed permeabilized (for iERalpha) vs. nonpermeabilized cells (for mERalpha). Expression of mERalpha was highest at low cell densities (<1000 cells/well) and decreased significantly at densities where cellular processes touched, whereas the more abundant iERalpha increased with increasing cell density over the same range. Serum starvation for 48 h caused increases in mERalpha, whereas iERalpha levels showed no significant changes. A large decline in mERalpha and iERalpha levels with cell passage number was observed. Minutes after nM 17beta-estradiol (E2) treatment, a portion of the cells rounded up and detached from the culture plate, whereas nM cholesterol had no such effect. Although E2 treatment did not change mERalpha levels, the antigen was reorganized from a fine particulate to aggregation into asymmetric large granules of staining. That common culturing conditions favor down-regulation of mERalpha may explain the relatively few reports of this protein in other experimental systems.
我们采用改良的免疫细胞化学条件，对大鼠垂体肿瘤细胞系GH3/B6/F10中的膜形式雌激素受体α（mERα）进行定量分析。我们使用96孔板免疫分析法研究了mERα与细胞内雌激素受体α（iERα）水平之间的调控关系。抗雌激素受体α抗体C542用于标记固定通透细胞（用于检测iERα）和非通透细胞（用于检测mERα）中的雌激素受体α（通过共轭碱性磷酸酶）。mERα的表达在低细胞密度（<1000个细胞/孔）时最高，在细胞突起相互接触的密度下显著降低，而含量较多的iERα在相同范围内随细胞密度增加而增加。血清饥饿48小时导致mERα增加，而iERα水平无显著变化。观察到mERα和iERα水平随细胞传代次数大幅下降。用纳摩尔浓度的17β-雌二醇（E2）处理数分钟后，部分细胞变圆并从培养板上脱落下来 . 而纳摩尔浓度的胆固醇则无此作用。虽然E2处理并未改变mERα水平，但抗原从细微颗粒重新组织为聚集成不对称的大染色颗粒。常见的培养条件有利于mERα的下调，这可能解释了在其他实验系统中关于这种蛋白质的报道相对较少的原因。
