Jin Pu

Apical-basal polarity in epithelial cells is a fundamental process in the morphogenesis of many tissues. But how epithelial cells become oriented with functionally specialized luminal and serosal facing membranes is not understood fully. Cell-cell and cell-substrate contacts induce the asymmetric distribution of Na⁺/K⁺-ATPase pumps on basal membrane and are essential for apical-basal polarity formation. Inhibition of the Na⁺/K⁺-ATPase pump abolished apical formation completely. But it is unclear how this pump regulated the apical polarity. We discovered that the transepithelial potential difference (TEP) which is dependent on the basal Na⁺/K⁺-ATPase distribution acts as an essential coordinating signal for apical membrane formation through Ror2/ERK1/2/LKB1 signaling. A similar concept applies to all other ion-transporting epithelial and endothelial tissues and this raises the possibility of regulating the TEP as a therapeutic intervention for disorders in which epithelial function is compromised by faulty electrical signaling.

BACKGROUND: Chloride channel accessory 1 (CLCA1) is a CLCA protein that plays a functional role in regulating the differentiation and proliferation of colorectal cancer (CRC) cells. Here we investigated the relationship between the level of CLCA1 and the prognosis of CRC.METHODS: First, the level of CLCA1 was detected quantitatively in normal and cancerous colonic epithelial tissues with immunohistochemistry. Next, the correlations between CLCA1 expression, pathological tumor features, and the overall survival rate of patients was analyzed. Finally, 3 publicly available data sets from the Gene Expression Omnibus were examined: normal CRC versus early CRC (GSE4107), primary CRC versus metastatic lesions (GSE28702), and low chromosomal instability versus high chromosomal instability (GSE30540).RESULTS: The expression of CLCA1 was decreased markedly in tumor specimens. CLCA1 expression was correlated significantly with the histological grade (P < .01) and lymph node metastasis (P < .01). A significantly poorer overall survival rate was found in patients with low levels of CLCA1 expression versus those with high expression levels (P < .05). The results confirmed that the low expression of CLCA1 in CRC was highly associated with tumorigenesis, metastasis, and high chromosomal instability. In addition, the loss of CLCA1 disrupted the differentiation of human colon adenocarcinoma cells (Caco‐2) in vitro.CONCLUSIONS: These findings suggest that CLCA1 levels may be a potential predictor of prognosis in primary human CRC. Low expression of CLCA1 predicts disease recurrence and lower survival, and this has implications for the selection of patients most likely to need and benefit from adjuvant chemotherapy. Cancer 2015;121:1570–1580. © 2015 American Cancer Society.

The apicobasal polarity of enterocytes determines where the brush border membrane (apical membrane) will form, but how this apical membrane faces the lumen is not well understood. The electrical signal across the epithelium could serve as a coordinating cue, orienting and polarizing enterocytes. Here we show that applying a physiological electric field (EF) to intestinal epithelial cells, to mimic the natural EF created by the transepithelial potential difference, directed phosphorylation of the actin-binding protein ezrin, increased expression of intestinal alkaline phosphatase (ALPI, a differentiation marker) and remodelled the actin cytoskeleton selectively on the cathode side. In addition, an applied EF also activated ERK1/2 and LKB1, key molecules in apical membrane formation. Disruption of the tyrosine-protein kinase transmembrane receptor Ror2 suppressed activation of ERK1/2 and LKB1 significantly and subsequently inhibited apical membrane formation in enterocytes. Our findings indicate that the endogenous EF created by the TEP may act as an essential coordinating signal for apical membrane formation at a tissue level, through activation of LKB1 mediated by Ror2/ERK signalling.

Breaking the balance between proliferation and differentiation in animal cells can lead to cancer, but the mechanisms maintaining this balance remain largely undefined. The calcium activated chloride channel A1 (CLCA1) is a member of the calcium sensitive chloride conductance family of proteins and is expressed mainly in the colon, small intestine and appendix. We show that CLCA1 plays a functional role in differentiation and proliferation of Caco-2 cells and of intestinal tissue. Caco-2 cells spontaneously differentiate either in confluent culture or when treated with butyrate, a molecule present naturally in the diet. Here, we compared CLCA1 expressional levels between patients with and without colorectal cancer (CRC) and determined the functional role of CLCA1 in differentiation and proliferation of Caco-2 cells. We showed that: 1) CLCA1 and CLCA4 expression were down-regulated significantly in CRC patients; 2) CLCA1 expression was up-regulated in Caco-2 cells induced to differentiate by confluent culture or by treatment with sodium butyrate (NaBT); 3) Knockdown of CLCA1 with siRNA significantly inhibited cell differentiation and promoted cell proliferation in Caco-2 confluent cultures, and 4) In Caco-2 3D culture, suppression of CLCA1 significantly increased cell proliferation and compromised NaBT-induced inhibition of proliferation. In conclusion, CLCA1 may contribute to promoting spontaneous differentiation and reducing proliferation of Caco-2 cells and may be a target of NaBT-induced inhibition of proliferation and therefore a potential diagnostic marker for CRC prognosis.

Neural induction is largely considered a default process, whereas little is known about intrinsic factors that drive neural differentiation. Kamiya and colleagues now demonstrate that a transcription factor, Zfp521, is capable of directing embryonic stem (ES) cells into neural progenitors. They discovered that Zfp521 transcripts were enriched in early neural lineage of ES cell differentiation. Forced expression of Zfp521 turned ES cells into neural progenitors in culture conditions that would normally inhibit neural differentiation. Zfp521 was expressed in mouse embryos during gastrulation. The protein was shown to associate with a co-activator p300 and directly induce expression of early neural genes. Knockdown of the Zfp521 by shRNA halted cells at the epiblast stage and suppressed neural differentiation. Zfp521 is a nuclear protein with 30 Krüppel-like zinc fingers mediating multiple protein-protein interactions, and regulates transcription in diverse tissues and organs. The protein promotes proliferation, delays differentiation and reduces apoptosis. The findings by Kamiya and colleagues that Zfp521 directs and sustains early neural differentiation now opens up a series of studies to investigate roles of Zfp521 in stem cells and brain development of mice and men.

Endogenous electrical fields (EFs) at corneal and skin wounds send a powerful signal that directs cell migration during wound healing. This signal therefore may serve as a fundamental regulator directing cell polarization and migration. Very little is known of the intracellular and molecular mechanisms that mediate EF-induced cell polarization and migration. Here, we report that Chinese hamster ovary (CHO) cells show robust directional polarization and migration in a physiological EF (0.3–1 V/cm) in both dissociated cell culture and monolayer culture. An EF of 0.6 V/cm completely abolished cell migration into wounds in monolayer culture. An EF of higher strength (≥1 V/cm) is an overriding guidance cue for cell migration. Application of EF induced quick phosphorylation of glycogen synthase kinase 3β (GSK-3β) which reached a peak as early as 3 min in an EF. Inhibition of protein kinase C (PKC) significantly reduced EF-induced directedness of cell migration initially (in 1–2 h). Inhibition of GSK-3β completely abolished EF-induced GA polarization and significantly inhibited the directional cell migration, but at a later time (2–3 h in an EF). Those results suggest that GSK-3β is essential for physiological EF-induced Golgi apparatus (GA) polarization and optimal electrotactic cell migration.