Regulation and role of brush border-associated ERK1/2 in intestinal epithelial cells Marie-Josée Boucher and Nathalie Rivard
CIHR Group on Functional Development and Physiopathology of the Digestive Tract, Département d’Anatomie et Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, QC, CanadaReceived 17 September 2003. Available online 8 October 2003. Abstract We have recently shown that elevated extracellular signal-regulated kinase (ERK) activities stimulate proliferation of intestinal cells whereas 然而 low sustained levels of ERK activities correlate with 相互关联 Gl arrest 阻止 and are required for expression of several enterocyte 肠囊肿 differentiation proteins. In an attempt to clarify how ERK1/2 regulates intestinal differentiation, the present study assessed the subcellular distribution and regulation of ERK proteins and activities in differentiated enterocytes. We report that (1) ERK1/2 and their upstream modulators Ras, p85 (PI-3K), Rac1, and MEK1 are found in the brush border; (2) brush border-associated ERK1/2 are stimulated by EGF and feeding; (3) immunoblotting of proteins phosphorylated on SP/K motif suggests the presence of ERK substrates in the brush border, one of which could be actin; and (4) pharmacological inhibition of ERK alters microvilli architecture. Our results suggest that ERK may play important roles in the control of microvilli structure and possibly, in brush border-associated responses in differentiated intestinal epithelial cells. 我们近期报道增强细胞间 ERK 活性刺激小肠细胞的增殖，然而较低的 ERK 活性水平与葡萄糖阻止相互关联，它 们也是一些肠细胞分化蛋白质表达所需要的。在一次尝试阐明 ERK1/2 怎样调节小肠分化中，现有的研究估计在分化的 肠细胞 ERK 蛋白质类和活动的亚细胞进行分布和调节。我们曾报道过：1、erk1/2 和它们的上游调控因子 PI-3K、Rac1 和 MEK1 在刷状缘膜发现。2、刷状缘膜相关的 ERK1/2 可以被 EGF 和采食刺激。3、在 SP/K 基序上的蛋白质免疫印迹磷酸化显 示 ERK 在刷状缘膜上的一个底物可能是肌动蛋白。4、药理学抑制 ERK 改变微绒毛结构。我们的结果显示 ERK 可能在微绒 毛的结构和刷状缘膜相关的小肠上皮细胞分化反应中起重要作用。 Author Keywords:
ERK ; Brush border; Junctions; Intestinal epithelial cell differentiation
Adult small intestinal enterocytes are highly polarized columnar cells with structural components adapted to the digestive function. These epithelial cells are endowed with a highly ordered apical brush border specifically designed for providing the enterocyte with maximal absorptive capacity for nutrient assimilation. Each of the 1000–2000 microvilli of a fully mature brush border is supported by a bundle of actin filaments, the basal end of which descends into the apical cytoplasm forming a rootlet approximately 1 μM below the membrane. Here, a dense and complex meshwork of filaments in this so-called terminal web region interdigitates between the rootlets serving most likely to stabilize the overall brush border architecture while conferring a contractile capacity, modulating transepithelial resistance and nutrient transport [1 and 2]. 成熟小肠细胞是高度极化的柱状细胞，结构构成由消化功能调节。这些上皮细胞被赋予用一个高端的绒毛顶端 刷状缘膜，它是特殊设计的，为了使肠细胞以最大吸收能力进行养分同化。每 1000-2000 高度成熟的微绒毛被一束肌 动蛋白丝支持，它们下降到顶端细胞质形成小跟大约在细胞膜下 1um。在这里，密集而复杂的网状组织丝在这个被称为 终末网区域在小跟当与一种收缩能力对照时最可能用于固定全部的刷状缘膜结构和调节经上皮抵制、养分运输相互交 叉。 Intestinal epithelial cells are continuously exposed to various stimuli such as bacteria and associated products (endotoxins), dietary components, luminal growth factors, and inflammatory mediators (cytokines). Enterocytes react to these stimuli by rapidly adjusting the microvillus length, and thus their absorptive capacity, in response to changing needs incurred by gut activity during digestion and absorption of various dietary nutrients [3, 4 and 5]. 小肠上皮细胞持续暴露于各种各样的刺激，如细菌和相关的制品（内霉素），饮食成分，苯巴比妥类生长因子 和炎症介质。肠细胞通过快速调节微绒毛长度应答这些刺激，这样他们吸收能力适应由肠活性在消化和吸收各种各样 的饮食营养导致的不断变化的需要。 The response of intracellular signaling pathways within the intestine to septic insult has been studied extensively. Binding of endotoxin, growth factors or cytokines to membrane receptors activates a series of intracellular signaling cascades, including families of several PKC isozymes and MAP
kinases such as extracellular signal-regulated kinase ( ERK) , Jun amino-terminal kinase (JNK), and p38, ultimately resulting in various gene expression and biological responses . These mechanisms are designed to allow the enterocyte to maintain intestinal homeostasis. 胞内信号通路的反应在肠腐败损伤内被广泛的研究过。内毒素结合，生长因子或膜受体细胞因子类活化 一系列细胞内的信号级联放大，包括一些 PKC 同功酶和 MAP 激酶（例如细胞外信号调节激酶 ERK、JNK 和 P38）家族，最终导致不同的基因表达和生物学反应。这些机制被设计来保持肠细胞保持肠细胞稳态。 To achieve the specificity of each signaling pathway, cells often employ compartmentalization. Information as to the compartmentalization of specific signaling on microvilli of intestinal epithelial cells remains limited. Phosphatidylinositol 3-kinase (PI-3K) , cPLA2 , and myosin light chain kinase (MLCK) [2 and 7] have been reported to be localized at the apical surface or within the brush border of enterocytes. In the present study, we demonstrate that the ERK1/2 are localized in the brush border and may act locally to modulate microvilli architecture and brush border-associated function in human enterocytes.
为了完成每个信号通路的专一性，细胞常利用区室化。小肠上皮细胞微绒毛上的关于区室化的特异信号肽的信息 还很有限。PI-3K（磷酸酯酰肌醇 3 激酶）、cPLA2 和肌球蛋白轻链激酶 MLCK 被报道位于顶端的表面或者在肠细胞的刷 状缘膜。在近期的研究中，我们证实 ERK1/2 位于刷状缘膜，也可能局部作用于调节微绒毛结构和刷状缘膜相关的功能。 Materials and methods
Materials. Monoclonal antibody HIS-14 against sucrase-isomaltase was provided by Dr A. Quaroni (Cornell University, Ithaca, NY, USA). Antibody recognizing the Na+/K+ ATPase pump was a kind gift from Dr. D.M. Fambrough (Johns Hopkins University, Baltimore, MD, USA). Dr. J. Pouysségur (University of Nice, France) provided the rabbit antibody that specifically recognizes MEK1. Antibodies raised against villin, E-cadherin, Rac1, and PKCα were purchased from Transduction Laboratories (Mississauga, Ont., Canada). Antibody directed against the p85 regulatory subunit of PI-3K was obtained from Upstate Biotechnology (Lake Placid, NY, USA). Anti-AKT and anti-phospho- ERK were purchased from Cell Signaling (Mississauga, Ont., Canada) and anti-ERK1 (C16) was from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Antiserum OP40 specifically recognizing p21Ras was from Oncogene Sciences (Calbiochem, Mississauga, Ont., Canada). The antibody specifically recognizing phosphoserine with a proline or lysine at their C-terminal was purchased from Biomol Cedarlane Laboratories (Hornby, Ont., Canada). EGF was from Collaborative Biomedical (Bedford, MA, USA). TNFα, IL1β, and IL6 were purchased from R&D Systems (Minneapolis, MN, USA). All other materials were obtained from Sigma–Aldrich (Oakville, Ont., Canada) unless stated otherwise. 单克隆抗体 HIS-14 蔗糖酶-异麦芽糖酶由 Dr A. Quaroni 提供。抗体识别钠钾 ATP 酶泵是 Dr. D.M. Fambrough 的 一种能力。Dr. J. Pouysségur 提供可以被 MEK1 特异识别的兔子抗体。绒毛蛋白，钙黏着蛋白，Rac1 和 PKCa（蛋白激 酶 c）的抗体从 Transduction Laboratories 购买。PI-3K 的 P85 调节亚基抗体从 Upstate Biotechnology 获得。AKT 和 ERK 的抗体从 Santa Cruz Biotechnologies 获得。特异性识别 P21Ras 的抗血清 OP40 从 Oncogene Sciences 获得。特异 性在 C 端识别有脯氨酸或赖氨酸磷酸丝氨酸的抗体从 Biomol Cedarlane Laboratories 获得。TNFα, IL1β, and IL6 从 R&D Systems 购买。其它用品从 Sigma 获得，除非另有说明。
Animals. Sprague–Dawley male rats (170–190 g) were purchased from Charles River Laboratories (St-Constant, QC, Canada). Animals were fed Purina chow and water ad libitum until the start of the experiments and kept in a controlled temperature and light cycle environment (21 °C; 12 h light, 12 h darkness). All studies were conducted in agreement with the principles and procedures outlined in the Canadian Guidelines for Care and Use of Experimental Animals. Eight rats were fasted during 18 h. Thereafter, 4 fasted rats were sacrificed by decapitation. Of the remaining 4 fasting rats, 2 were refed for 1 h and two others re-fed for 3 h. The animals were then sacrificed and the jejunum was removed for preparation of brush border membrane vesicles (BBMV), as described below. Human tissues and cell culture. Tissues from human fetuses varying in age from 18 to 20 weeks of gestation (post-fertilization fetal ages estimated according to Streeter ) were obtained from normal elective pregnancy terminations. No tissue was collected from cases associated with a known fetal abnormality or fetal death. Studies were approved by the Institutional Human Subject Review Board (University of Sherbrooke, Sherbrooke, QC, Canada). The human adenocarcinoma Caco-2/15 cell line was obtained from A. Quaroni (Cornell University, Ithaca, NY, USA) and cultured as previously
described [9 and 10]. These cells undergo morphological and functional differentiation to an enterocyte phenotype several days after they have reached confluence [9 and 10]. 人体组织和细胞培养：从怀孕期 18 到 22 周人体胎儿组织被获得从正常选择的妊娠终端。
Preparation of brush border membrane vesicles. The mucosae of rat jejunum and human fetal jejunum were scraped and homogenized (HO) or differentiated Caco-2/15 cells (at least 25 days postconfluent) were lysed (HO) in Tris–Hepes 2 mM containing 50 mM mannitol, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 10 μg/ml leupeptin, 1 μg/ml pepstatin, and 10 μg/ml aprotinin. BBMV were prepared by the calcium chloride precipitation method as previously described . CaCl2 was added to a final concentration of 10 mM for 10 min. Centrifugation of the Ca2+-treated homogenate at 5000g for 15 min yielded a P1 fraction which contains the total recovered DNA and more than 90% of mitochondrial, microsomal, and basolateral membranes. The P1 fraction was then resuspended in Laemmli’s buffer (62.5 mM Tris–HCl, pH 6.8, 2.3% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.005% bromophenol blue, and 1 mM PMSF). The first supernatant (S1) was centrifuged at 30,000g for 30 min. The second supernatant (S2) which contains lysosomes and some microsomal membranes and the second pellet (P2) which contains BBMV were then lysed in Laemmli’s buffer.
刷状缘膜囊的准备：鼠空肠粘膜和人胎儿空肠经过冲洗和经过均匀加工处理或者分化型 Caco-2/15 细胞（至少 25 天融合后）被在 Tris–Hepes（三羟基甲基氨基）2mmol 融合包括 50mmol 甘露醇，0.1mmolPMSF 苯甲基磺酰氟化物， 10ug/ml 亮肽酶素，1ug/ml 胃蛋白酶抑制剂，10ug/ml 抑肽酶。BBMV（刷状缘膜囊）用前面提到过的氯化钙沉淀法获 得。
Western blotting. Western blot analyses were performed essentially as described previously . Immunofluorescence. Fully differentiated Caco-2/15 cells (30 days post-confluence) were embedded in optimum cutting temperature compound, and quickly frozen in liquid nitrogen. Frozen sections of 3 μm were spread onto silane-coated glass slides and air-dried. Samples were immunostained for 1 h with primary antibody and 30 min with secondary antibody. Negative controls (no primary antibody) were included in all experiments Negative for metastatic neoplasm 荧光免疫检验法：完全分化的 Caco-2/15 细胞（30 天会和后）在最适合切割的温度被嵌入，并快速冷冻在液氮 中。冷冻切片 3um 在甲硅烷覆盖的载玻片上展开，风干。样品用免疫荧光染色法用主要抗体 1h 和次要抗体 30min。所有 的实验都有负控制。忽略肿瘤迁移。
Electron microscopy. Electron microscopy was performed as described previously . All reagents were purchased from Electron Microscopy Sciences (Cedarlane, Hornby, Ont., Canada). 电子显微镜检查：
Isolation of cytoskeleton-associated proteins. Cells were first washed twice with ice-cold PBS and then soluble proteins were extracted on ice with cold lysis/cytoskeleton stabilization buffer (0.5% Triton X-100, 50 mM NaCl, 10 mM PIPES, pH 6.8, 300 mM sucrose, and 3 mM MgCl2). Cytoskeletonassociated proteins (insoluble fraction) were harvested by centrifugation (13,000 rpm at 4 °C for 10 min) and solubilized in Laemmli’s buffer. Finally, ERK and E-cadherin levels were determined by immunoblot of the cytoskeleton and soluble fractions. 分离细胞骨架上的蛋白质类。细胞首先在冰 PBS 中冲洗 2 次，然后可溶性蛋白在冰上用冰容菌/细胞骨架稳定缓 冲液（0.5% Triton X-100, 50 mM NaCl, 10 mM PIPES, pH 6.8, 300 mM sucrose, and 3 mM MgCl2）提取。细胞骨架 上的蛋白质用离心(13,000 rpm at 4 °C for 10 min)，在电泳示踪缓冲液溶解。最后，ERk 和 E-cadherin 水平用细 胞骨架和可溶性分数的免疫印迹来确定。 Results Subcellular distribution of ERK1/2 and other signaling proteins in differentiated enterocytes 在分化的肠细胞中的 ERK1/2 亚细胞分布和信号蛋白 In an attempt to analyze the subcellular distribution of ERK proteins in differentiated enterocytes, homogenates from intestinal fetal mucosae and from 25-day post-confluent Caco-2/15 cells were fractionated and brush border vesicles were prepared by Ca 2+ precipitation, as described
previously . As expected, the brush border-enriched fraction (P2) was found to be enriched in villin and sucrase-isomaltase while the P1 fraction contained the bulk of the basolateral membranes as visualized by protein expression of the Na+/K+ ATPase pump (Fig. 1A). E-cadherin was distributed throughout the P1 and P2 fractions, consistent with previous observation that E-cadherin is expressed along the lateral membrane of intestinal epithelial cells . As shown in Fig. 1B, expression of ERK1 and ERK2 was detected in all fractions including the brush border-enriched P2 fraction. Using a polyclonal antibody specific for the detection of phosphorylated ERK1 and ERK2, ERK activity was mostly detected in the P1 and P2 fractions derived from the human fetal intestinal mucosae but uniformly detected in all fractions derived from differentiated Caco-2/15 cells. On longitudinal sections, immunostaining of differentiated Caco-2/15 cells (30 days post-confluence) confirmed that ERK1/2 were distributed uniformly. Interestingly, a sharp staining of the apical surface of the epithelial cells was also observed ( Fig. 1C, see arrowheads). As expected, sucrase-isomaltase, an enterocyte differentiation marker, was detected at the apex of the cells ( Fig. 1C, see arrowheads).
在一次尝试分析 ERK 蛋白在分化型肠细胞的亚细胞分布中，从胎儿小肠粘膜和融合 25 天后 Caco-2/15 的细胞被 分割，刷状缘小囊泡用钙沉淀法准备好。与预期相同，P2（富含有刷状缘部分）富含绒毛蛋白和蔗糖酶-异麦芽糖酶当 P1 部分包括由钠钾 ATP 酶泵的蛋白质表达的直观的大量的基地外侧膜。E-cadherin 分布在 P1 和 P2 中，与前期观测到 的 E-cadherin 在小肠上皮细胞侧膜上的表达一致。在 Fig.1B 中显示 ERK1 和 ERk2 存在于所有的部分包括富含刷状缘膜 的 P2 部分。用特异性多克隆抗体检测磷酸化 ERK1 和 ERK2，ERK 活性主要在从人体胎儿小肠粘膜的 P1 和 P2 部分检测到， 但在从分化型 Caco-2/15 cells 中的各部分均匀的检测到。在纵切面，免疫染色分化型 Caco-2/15 cells 确认 ERK/2 分 布均匀。有趣的是，上皮细胞顶端表面观测到清晰的染色。从研究中得知，蔗糖酶-异麦芽糖酶是肠细胞分化标志， 被在细胞的顶点发现。 Full-size image (31K)
Fig. 1. Subcellular distribution of ERK1/2 and other signaling proteins in differentiated enterocytes. BBMV were prepared from mucosae of human fetal intestine or 25-day post-confluent Caco2/15 cells, as described in Materials and methods. Equal amounts of proteins from each fraction were separated by SDS–PAGE and protein expression was analyzed by Western blotting using antibodies against sucrase-isomaltase, villin, Na+/K+ ATPase, and E-cadherin (A) or ERK1/2 and phosphorylated ERK1/2 (B) or Ras, MEK1, p85/PI-3K, AKT, Rac1, and PKCα (D). H0, homogenate; S1, supernatant 1; P1, pellet 1; S2, supernatant 2; P2, and pellet 2 containing BBMV. P- ERK, phosphorylated and activated ERK. (C) Fully differentiated Caco-2/15 cells were embedded in OCT freezing medium and sectioned longitudinally, fixed, and labeled for ERK1/2 and sucrase-isomaltase proteins.
ERK1/2 和其它信号蛋白在分化型肠细胞中的亚细胞分布。BBMV 用从人体胎儿小肠或 25 天 post-confluent Caco2/15 cells 上的粘膜准备好。从各部分获得等量的蛋白质用 SDS–PAGE 分离，蛋白质表达用 western blotting 分析， western blotting 用 sucrase-isomaltase, villin, Na+/K+ ATPase,和 E-cadherin (A) or ERK1/2 和磷酸化 ERK1/2 (B) or Ras, MEK1, p85/PI-3K, AKT, Rac1, and PKCα(D)的抗体。HO、匀浆；S1、上清液；P1、小片；S2、上清液； P2，和小片 2 包括 BBMV。P-ERK，磷酸化和有活性的 ERK。完全分化的 Caco-2/15 cells 植入到 OCT 冷冻介质和纵切面， 固定，和标记 ERK1/2 和蔗糖酶-异麦芽糖酶蛋白。 The compartmentalization of ERK activity in several fractions and particularly in the brush border fraction prompted us to investigate whether known upstream regulators of ERK cascade were also expressed in these fractions in both human fetal intestine and differentiated Caco-2/15 cells. The expression of Ras, MEK1, the p85 regulatory subunit of PI-3K, Akt, Rac1, and PKCα, all known to potently influence the ERK signaling cascade in different cell types, was therefore analyzed . As shown in Fig. 1D, the small G proteins Ras and Rac as well as p85 (PI-3K) and PKCα were mostly detected in the P1 and P2 fractions from both fetal intestine and differentiated Caco-2/15 cells. The dual specificity kinase MEK1 and the kinase Akt were also detected in the P1 and P2 fractions but were predominantly found in the supernatant fractions. ERk 活性的区室作用在一些部分尤其是刷状缘部分提示我们研究，已知的 ERK 的上游调节因子级联在人体胎儿小 肠和分化型 Caco-2/15 cells 中是否也表达。 Ras, MEK1, PI-3K 的调节亚单位 p85, Akt, Rac1, and PKCα 的表达已
知可以有力的影响在不同细胞类型中 ERK 信号级联放大，因此，我们研究它们。如 Fig。1D 所示，小 G 蛋白 Ras 和 Rac 以及 P85（pi-3k）和 PKCa 主要在胎儿小肠和分化型 Caco-2/15 细胞的 P1 和 P2 区域被发现。双特异性激酶 MEK1 和激酶 Akt 也在 P1 和 P2 区域被发现，但主要在上清液中发现。 Effect of EGF, TNF α, IL-6, IL-1β, and feeding on ERK1/2 activity in enterocytes In an attempt to characterize the regulation of ERK1/2 in differentiated intestinal epithelial cells, enzymatic activation of ERK1/2 by various agonists was assessed. Cell fractions were prepared from 3 week-post-confluent cells initially stimulated for 15 min with EGF (50 ng/ml) and for 20 min with TNF-α (30 pg/ml), IL-6 (10 pg/ml) or IL-1β (10 ng/ml). As shown in Fig. 2A, control differentiated Caco-2/15 cells exhibited minimal ERK activities in all recovered fractions. Addition of EGF dramatically increased ERK activities in all fractions, including the brush border-enriched fraction (P2). By contrast, treatment with TNF-α, IL-6 or IL-lβ enhanced ERK1/2 phosphorylation levels, moderately in S1 and S2 fractions and slightly in P1 and P2 fractions. These modulations occurred without any variation in total amount or subcellular distribution of ERK -1/2 (Fig. 2B). There was no significant induction of ERK activities detected in any of the fractions following 20 min of LPS or butyrate treatment (data not shown). 在一次尝试描述 ERK/2 在分化型的小肠上皮细胞的调节中，ERK1/2 的酶活性被评估了。细胞部分用 3 周 postconfluent 细胞最初用 EGF(50 ng/ml)处理 15 分钟，再用 NF-α (30 pg/ml), IL-6 (10 pg/ml) or IL-1β (10 ng/ml)处理 20 分钟。如 Fig。2A,控制分化型 Caco-2/15 细胞在所有回复部分显示最小 ERK 活性。添加 EGF 显著增加 ERK 在所有部分的活性，包括 P2。相反的是，TNF-α, IL-6 or IL-lβ 适度加强了 ERK1/2 在 S1 和 S2 部分的磷酸化水 平，P1 和 P2 区域少量增加。这些调节产生没有在数量和 ERK 的亚细胞分布上出现任何不同。在 20 分钟 LPS 处理和丁酸 盐处理的任何区域都没有发现显著的 ERK 活性差异。
Fig. 2. Effect of EGF, TNFα, IL-6, IL-1β, and feeding on ERK1/2 activity in enterocytes. (A) Twenty day post-confluent Caco-2/15 cells seeded on a Transwell polycarbonate filter were stimulated basolaterally with EGF (50 ng/ml) for 15 min. (B) Twenty-five-day post-confluent Caco-2/15 cells were untreated (control) or treated for 20 min with TNF-α (30 pg/ml), IL-6 (10 pg/ml) or IL-lβ (10 ng/ml). (C) Rats were sacrificed by decapitation after 18 h of starvation or after 1 and 3 h of re-feeding. (A–C) BBMV were prepared, equal amounts of proteins from each fraction were separated by SDS–PAGE, and protein expression was analyzed by Western blotting using antibodies against ERK1/2 and phosphorylated ERK1/2 (P- ERK) . Fig。2. 小肠细胞中 EGF, TNFα, IL-6, IL-1β, 和采食对 ERK1/2 的活性的影响。（A）20 天 post-confluent Caco-2/15 细胞 seeded on a Transwell polycarbonate filter 在底外侧用 EGF（50ng/ml）处理 15 分钟。(B)25 天 post-confluent Caco-2/15 细胞不经处理和经过 TNF-α (30 pg/ml), IL-6 (10 pg/ml) or IL-lβ (10 ng/ml)处理 20 分钟。(c)小鼠在禁饲 18 小时后和饲喂 1h 和 3h 后被断头杀死。(A–C) BBMV 准备好，各个部分等量的蛋白质用 SDSPAGE 分离，用 ERK 和磷酸化 ERK1/2 的抗体用 Western blotting 分析蛋白质表达。 As the primary function of the intestinal epithelium is the terminal digestion and absorption of water and nutrients, ERK1/2 activities were analyzed in rat gut epithelium under conditions of fasting and of re-feeding. Rats were fasted overnight and re-fed for 1 or 3 h. One hour after feeding, pancreatic enzymes and biliary acid have now reached the duodenum, initiating the process of terminally digesting the chyme emanating from the stomach. After 3 h of feeding, digestive and absorptive processes are fully initiated . In rat intestinal mucosae, total and phosphorylated ERK1/2 were mostly detected in P1 and P2 fractions ( Fig. 2C). Re-feeding of fasted rats markedly enhanced ERK phosphorylation levels, especially after 3 h when terminal digestion and absorption
were well initiated . These modulations occurred without any variation in total amount or subcellular distribution of ERK -1/2 ( Fig. 2C). Of note is that the highest ERK1/2 activation levels were observed in BBMV fractions (P2) suggesting that, in a physiological context of normal differentiated enterocytes, ERK1/2 proteins may regulate brush border-associated functions.
消化和吸收水分和营养是小肠上皮细胞的主要功能，分析 ERK 活性在小鼠肠上皮在禁饲和再饲喂处理后。小鼠在 禁饲后再饲喂 1h 和 3h。饲喂 1h 后，胰酶和胆酸到达十二指肠，开始了消化从胃出来的食糜的过程。饲喂 3h 后，消化 和吸收过程全面开始了。在小鼠小肠粘膜，总的和磷酸化的 ERK1/2 主要在 P1 和 P2 检测到。禁饲的小鼠再饲喂后显著加 强了 ERK 的磷酸化水平，特别是 3h 后消化和吸收完全开始时。这些调节发生时没有任何 ERK 总量和亚细胞分布的差别。 在 BBMV 中发现最高的 ERk 活性水平显示，在正常的分化型肠细胞的生理环境中，ERK1/2 蛋白可能有调节刷状缘相关 的功能。 Evidences of ERK1/2 substrates in brush border of enterocytes In a first attempt to identify brush border-associated ERK substrates, differentiated Caco-2/15 cells were treated with UO126, followed by preparation of brush border-enriched fractions, and by an immunoblot with an antibody which specifically recognizes proteins phosphorylated on a serine localized immediately ahead of either a proline or lysine. ERK1/2 are proline-directed serine/threonine kinases recognizing the consensus sequence PX(T/S)P . As shown in Fig. 3A, treatment of differentiated Caco-2/15 cells with UO126 drastically inhibited ERK1/2 activities in all fractions studied. Immunoblotting with phosphoserine antibodies revealed that one S1-associated protein (lane 4) and several P2-associated proteins (lane 10) were less phosphorylated following UO126 treatment. UO126 reduced phosphorylation of P2-associated proteins with approximate molecular masses of 31, 36, 46, and 48 kDa (Fig. 3A, lane 10, see arrows). When immunoblots were performed using the anti-actin antibodies, actin aligned precisely with p46 ( Fig. 3B). Therefore, these data reveal the presence of ERK substrates in the brush border of enterocytes, one of which could be actin.
在第一次尝试识别刷状缘相关 ERK 底物是，分化型 Caco-2/15 用 U0126 处理，然后把刷状缘富集部分准备好，然 后用免疫印迹，免疫印迹用可以特异性识别用丝氨酸在脯氨酸和赖氨酸之前立即集中的蛋白质。ERk 用脯氨酸定向，丝 氨酸-苏氨酸激酶识别共有序列 PX(T/S)P。如 Fig3A 显示，用 U0126 处理分化行 Caco-2/15 彻底抑制了 ERK 在所以研究 区域的活性。用磷酸丝氨酸的抗体的免疫印迹显示一个 S1 相关的蛋白和一些 P2 相关的蛋白用 U0126 处理后磷酸化减少。 U0126 在大约分子量 31、36、46 和 48KD 减少 P2 相关的磷酸化。当免疫印迹使用抗肌动蛋白抗体时，肌动蛋白精确的用 P46 对齐。因此，这些数据反应 ERK 底物在肠细胞刷状缘膜的表达量，肌动蛋白可能是一个底物。 Full-size image
Fig. 3. Evidences of ERK1/2 substrates in enterocyte brush border. (A,B) Twenty-five-day postconfluent Caco-2/15 cells were treated for 4 h with UO126 (10 μM) and BBMV were prepared as described in Materials and methods. Equal amounts of proteins from each fraction were separated by SDS–PAGE. Proteins phosphorylated on serine localized immediately ahead of either a proline or lysine as well as expression of ERK1/2 and phosphorylated ERK1/2 were analyzed by Western blotting. (B) Equal amounts of proteins from P2 fraction were separated by SDS–PAGE. Actin and proteins phosphorylated on serine localized immediately before either a proline or a lysine were analyzed by Western blotting. (C) Caco2/15 cells were harvested at subconfluence (day −2), confluence (day 0), and at day 3, 6, 9, and 12 of post-confluence. Cells were extracted on ice with cold lysis/cytoskeleton stabilization buffer. Soluble (S) and cytoskeleton-associated proteins (I) were separated by SDS–PAGE and subjected to immunoblotting for E-cadherin and ERK1/2.
ERK 底物在肠细胞刷状缘膜的证据。Twenty-five-day post-confluent Caco-2/15 细胞用 4 h with UO126 (10 μM)处理，BBMV 用前面提到的方法准备。从各部分获得的等量的蛋白质用 SDS-PAGE 分离。蛋白质磷酸化的丝氨酸。 从 P2 区域获得的等量蛋白质用 SDS-PAGE 分离。肌动蛋白和蛋白质磷酸化
The major components of the intestinal brush border involve actin cytoskeleton-associated proteins and hence cannot be extracted with a 0.5% solution of the non-ionic detergent Triton X-100, but are found in the insoluble fraction. Experiments were therefore conducted to analyze whether ERK1/2 are associated with the cytoskeleton in differentiating intestinal epithelial cells. As shown in Fig. 3C, differentiation of Caco-2/15 cells resulted in a significant increase in the proportion of E-cadherin associated with the cytoskeleton, correlating with the assembly of adherens junction components with cortical F-actin . Of special interest was the progressive increased association of ERK1 and ERK2 with the cytoskeleton during differentiation of Caco-2/15 cells, suggesting that ERK may regulate locally cytoskeletal-associated processes in enterocytes. 小肠刷状缘膜的主要成分包括：肌动蛋白、细胞骨架相关的蛋白质和因此不能用 0.5%非离子去污剂 Triton X100 的溶液提取但在不溶解的部分发现的。实验因此进行分析 ERK 是否与分化的肠上皮细胞的细胞骨架有关。如 Fig.3C 所示，分化型 Caco-2/15 细胞导致了 E-cadherin 与细胞骨架的比例显著增加，与皮质 F 肌动蛋白的黏结部组装有关。 有趣的是，这种改进增加了 ERK1 和 ERK2 与分化型 Caco-2/15 细胞的细胞骨架的联系，显示 ERK 可能调节肠细胞局部 的细胞骨架相关的过程。 Role of ERK1/2 in differentiated intestinal epithelial cells To investigate the role of ERK in intestinal epithelial cell differentiation, the impact of its inhibition was evaluated on morphological differentiation of Caco-2/15 cells. Cell cultures were characterized by transmission electron microscopy 4 days after confluence. As shown in Fig. 4, at 4 days post-confluence, Caco-2/15 cells began to exhibit certain ultrastructural characteristics similar to that found in the intact villus epithelium, including presence of microvilli (panel 2, see arrowheads) as well as junctional complexes (panel 2, see arrows). Addition of the pharmacological inhibitor of MEK1/2, UO126, at day 0 to 4 post-confluence did not alter cell polarization or the assembly and maturation of junctional complexes (panels 3 and 4, see arrows). This is consistent with the fact that inhibition of ERK in these cells did not influence transepithelial resistance (data not shown). However, several microvilli had an irregular, slightly swollen shape (Fig. 4, panel 4, see arrowheads) while others were fragmented, suggesting that ERK may be implicated in the control of microvilli architecture. 为了了解 ERK 在小肠上皮细胞分化中的作用，研究了抑制它对分化型 Caco-2/15 细胞的形态学的影响。细胞结构 用透射电子显微镜在集合 4 天后观察。在 Fig.4 中显示，在 post-confluence 的第四天 Caco-2/15 细胞开始显示出特定 的超微结构，特征与未受损的上皮细胞绒毛和微绒毛表面包括连接复合体都相似。加入的药理学抑制剂 MEK1/2、U0126 在 0 到 4 天 post-confluence 没有改变细胞极化以及连接复合体的组合和成熟度。这些与 ERK 抑制剂没有影响经上皮抵 制一致。然而，一些微绒毛有不规则、轻微的肿胀，一些变成了碎片，显示 ERK 可能参与控制微绒毛的形态。 Full-size image
Fig. 4. Role of ERK1/2 activity in differentiated intestinal cells. Caco-2/15 cells were treated from day 0 to day 4 of post-confluence with or without 10 μM UO126. Cells were fixed in glutaraldehyde and osmium tetroxide prior to epoxy embedding for electron microscopy analysis. Bars=4 μm for panels 1 and 3 and 333 nm for panels 2 and 4. ERK 在分化型小肠细胞中的活性。Caco-2/15 细胞用 0 to day 4 of post-confluence with or without 10 μM UO126 处理。细胞用戊二醛和四氧化锷固定前用 epoxy embedding 用与电子显微镜。 Discussion We have recently shown that elevated ERK activities stimulate proliferation of intestinal cells whereas low sustained levels of ERK activities correlate with G1 arrest and enterocyte differentiation. Addition of the MEK1 inhibitor, PD98059, during differentiation interfered with sustained activation of ERK and sucrase-isomaltase expression, suggesting a role of ERK signaling in differentiated intestinal epithelial cells . In a first attempt to clarify how ERK1/2 regulate
intestinal epithelial cell differentiation, the present study assessed the subcellular distribution of ERK proteins and activities in differentiated enterocytes. The prominent localization of ERK1/2 and their upstream modulators Ras, p85 (PI-3K), Rac1, and MEK1 in the brush border of human enterocytes suggests that one of the sites of action of ERK signaling is at the apex of these cells. This is consistent with the observation that inhibition of ERK alters microvillus structure and that stimulation of ERK activity by feeding occurs predominantly at the brush border of rat intestinal epithelial cells. Therefore, we propose that ERK may contribute to the regulation of some brush border-associated functions. 我们近期证明提高 ERK 的活性刺激小肠细胞的增殖，然而低含量水平的 ERK 活性与 G1 阻止和肠细胞分化相关。添 加 MEK1 抑制剂 PD98059 在分化期干扰 ERK 的活性和蔗糖酶-异麦芽糖酶的表达，显示 ERK 信号在小肠上皮细胞分化中 的作用。这是第一次尝试证实 ERK 是怎样调节小肠上皮细胞分化，现在的研究估计了 ERk 蛋白的亚细胞分布和在分化型 肠细胞中的活性。ERK 显著的局限化和它们上游调控因子 Ras、P85、Rac1、MEK1 在人肠细胞刷状缘膜显示一个 ERK 信号的 作用位点是这些细胞的顶点。这与观察到的 ERK 抑制剂改变了微绒毛的结构和采食主要在小肠水平细胞刷状缘刺激了 ERK 的活性的结果一致。因此，我们提出 ERK 可能调节一些刷状缘膜相关的功能。 The cellular localization of ERK in the brush border-associated enriched fraction of Caco-2 cells and intestinal epithelium places it at location where it can actively participate in apical signal transduction. In this regard, it is of interest that some of the known substrates of ERK1/2 have locations similar to those observed herewith for ERK proteins in human enterocytes. Among the potential substrates, cPLA2 has been localized at the apical surface of the plasma membrane and can be phosphorylated and activated by ERK1/2 . The MLCK present in the intestinal brush border [2 and 7] has been shown to be phosphorylated and activated by ERK1/2 in FG carcinoma cells . In addition, caldesmon and cortactin, two actin-binding proteins previously described as localized in the terminal web of enterocytes [15 and 16], are similarly phosphorylated by ERK1/2 [17 and 18]. This apical localization of ERK has also been reported in chicken  and human  intestinal epithelium. In addition, it has been recently reported that Ras, Raf, MEK, and ERK1/2 were all associated with microvillar microfilaments of ascite cells isolated from rat mammary adenocarcinoma . Therefore, the presence of ERK1/2 at a site involved in regulating microfilament and cell surface dynamics suggests that these enzymes may play important roles in brush border-associated responses or in the control of microvilli architecture.
ERK 在刷状缘膜相关的富含 Caco-2/15 细胞和小肠上皮细胞的细胞定位于它可以积极参与顶端信号传导的部分。 考虑到这些，有趣的是一些已知的 ERK 的底物与观察到的在人体肠细胞随同 ERK 的蛋白质的位置相似。在可能的底物中， cPA2 在质膜的顶端表面发现了，它可以被 ERK 磷酸化和激活。MLCK 存在于小肠刷状缘膜，显示在 FG 癌细胞中被 ERK 磷 酸化和激活。此外，钙介质素和 cortactin，两种肌动蛋白结合蛋白以前认为在肠细胞的终末网，同样可以被 ERK 磷酸 化。This apical localization ERK 在鸡和人的小肠上皮中被发现。此外，近期报道 Ras, Raf, MEK, and ERK1/2 与从 小鼠乳腺癌分离的 ascite cells 上的 microvillar microfilaments 相关。因此，ERK 在调节微丝和细胞表面动力学的 相关区域的出现表明这些酶类可能在刷状缘膜相关的反应和微绒毛的形态调节中有重要作用。 The brush border of intestinal epithelial cells, an evolutionary adaptation designed to increase the surface area for digestion and absorption of ions and nutrients, appears to be a primary site of action of EGF and gastrointestinal hormones in the small intestine [1 and 2]. For instance, EGF and diet have been shown to regulate transport function in differentiated intestinal cells [21 and 22]. This effect is associated with increases in both brush border surface area and total absorptive surface area, primarily due to an increase in microvillus height [22 and 23]. Of particular interest is the fact that ERK inhibition in enterocytes alters microvilli architecture. Indeed, we observed that many microvilli appeared somewhat swollen and even fragmented following treatment of cells with the MEK inhibitor. Such microvillus phenotype has been recently demonstrated in duodenal and jejunal villus epithelial cells in fasted rats . Interestingly, we found that feeding markedly enhanced brush border-associated ERK activities. Taken together these findings indicate that ERK1/2 may exert some biological effects via alterations in microvilli shape. This is consistent with our results demonstrating the localization of ERK1/2 proteins in the microfilament-rich apical region of enterocytes and their association with the cytoskeleton during differentiation. However, the mechanism by which ERK1/2 may alter microvilli structure remains to be determined. Our data do suggest however that brush border-associated proteins like actin could be phosphorylated on serine residues by ERK1/2 or in a ERK1/2-dependent manner. Such serine phosphorylation of actin has been reported following EGF
stimulation in A431 and HER14 cells . It has been suggested that a possible function of actin phosphorylation is to create a specific binding site for other proteins. Furthermore, there is evidence to suggest that actin phosphorylation facilitates actin polymerization . In addition an association of ERK with contractile filaments of actin has been previously reported in vascular smooth cells stimulated with phenylephrine . As such, it is plausible that the ERK -induced phosphorylation of actin is involved in increased actin polymerization observed in microvilli in response to EGF [3 and 23] or feeding [22 and 28]. It should be mentioned that caldesmon is another potential cytoskeletal target for brush border-associated ERK1/2. Indeed, caldesmon is a major actin-, myosin-, tropomyosin-, and calmodulin-binding protein which exhibits inhibitory function toward actintropomyosin-activated myosin ATPase activity. Phosphorylation of caldesmon by ERK1/2 has been shown to attenuate its interaction with actin  and to release actomyosin interaction . 小肠上皮细胞的 刷状缘膜是进化的调节，来增加消化和吸收营养的表面积，可能是小肠 EGF 和胃肠激素的主要作用位点。例 如，EGF 和饮食调节在分化型刷状缘膜表面区域和总吸收表面积区域的转运能力，主要是因为增加了微绒毛 的高度。有趣的是，ERK 抑制剂在肠细胞改变微绒毛的形态学结构。确实，我们观察到了一些微绒毛变得有些 肿胀和变成碎片在 MEK 抑制剂处理后。这样的微绒毛表型最近表明在十二指肠和空肠绒毛上皮细胞中发现。有 趣的是，我们发现饲喂明显加强了刷状缘膜表面的 ERK 活性。综合这些研究显示 ERK 可能在体内对微绒毛的 形态发生一些生物学影响。这与我们的结果显示 ERK 蛋白局限于肠细胞的 microfilament-rich apical 区域 和他们与细胞骨架在分化期相关。然而，ERK 可能改变微绒毛的结构的机制还有待确定。我们的数据支持刷状 缘膜相关蛋白如肌动蛋白可以用丝氨酸残基通过 ERK 或者一个 ERK 依赖的 manner 磷酸化。这样的肌动蛋白丝 氨酸磷酸化曾被报道用 EGF 刺激 A431 和 HER14 细胞后发现过。肌动蛋白磷酸化一个可能的功能是为 其它蛋白 建立一个特殊的结合位点。此外，有证据显示肌动蛋白磷酸化容易使肌动蛋白聚合。此外 ERK 和肌动蛋白有收 缩性的丝的联合以前被报道在 vascular smooth cells 用去氧肾上腺素刺激。像这样，ERK 诱导的肌动蛋白 磷酸化与观察到的微绒毛肌动蛋白聚合应答 EGF 或饲喂是可以的。需要提到的是，钙介质素是另一个可能的 brush border-associated ERK1/2 细胞支架目标。确实，钙介质素是一个主要的 actin-, myosin-, tropomyosin-, and calmodulin-binding 蛋白 exhibits inhibitory function toward actintropomyosin-activated myosin ATPase activity 。钙介质素通过 ERK 的磷酸化显示减弱了肌动蛋白和释 放了肌动球蛋白的相互作用。 In summary, our findings demonstrate that the ERK signaling pathway is present in the brush border of human intestinal epithelial cells. Moreover, ERK1/2 present in the brush border are stimulated by EGF and feeding, suggesting a role in nutrient, ion transport or other brush borderassociated functions. Our findings indicate that ERK1/2 may exert some biological effects via alterations in microvilli shape of enterocytes. However, a potential activation of these enzymes cannot be excluded following epithelial cell injury since ERK1/2 were significantly activated after mechanical wounding of IEC-6 cells . Current investigations are presently underway to further clarify the physiological role of brush border-associated ERK1/2 in differentiated intestinal epithelial cells.总的来说，我们的研究显示 ERK 信号通路在人小肠上皮细胞刷状缘膜是存在的。而且，ERK 存在于 刷状缘膜被 EGF 和饲喂刺激，显示在营养、离子吸收和其它刷状缘膜相关的功能。我们的研究显示 ERK 可能影响一些生 物学效果通过改变 肠细胞的微绒毛形状。然而，这些酶一个可能的激活作用不能排除上皮细胞的损伤因为 ERK 被显著 激活在 IEC-6 机械损伤后。现在的检查正在进行为了进一步阐明刷状缘膜相关的 ERK 在分化型小肠上皮细胞中的生理学 作用。 Acknowledgements We thank Pierre Pothier for a critical reading of the manuscript. Special thanks go to Dr Christiane Malo for her judicious comments and fruitful discussion in the course of this work. This research was supported by grant from Canadian Institutes of Health Research (MT-14405 to NR). N.R. is a recipient of a Canadian Research Chair in Signaling and Digestive Physiopathology. M.J.B. is a student scholar from the Fonds pour la Recherche en Santé du Québec. References 1. R. Holmes and R.W. Lobley, Intestinal brush border revisited. Gut 30 (1989), pp. 1667–1678. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (11) 2. D. Louvard, M. Kedinger and H.P. Hauri, The differentiating intestinal epithelial cell: establishment and maintenance of functions through interactions between cellular structures. Annu. Rev. Cell Biol. 8 (1992), pp. 157–195. View Record in Scopus | Cited By in Scopus (138)
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CIHR Group on Functional Development and Physiopathology of the Digestive Tract, Département d’Anatomie et Biologie Cellulaire, Faculté de M...