Expression of semaphorin 3A in the rat corneal epithelium during wound healing

The neural guidance protein semaphorin 3A (Sema3A) is expressed in corneal epithelial cells of the adult rat. We have now further investigated the localization of Sema3A in the normal rat corneal epithelium as well as changes in its expression pattern during wound healing after central corneal epithelial debridement. The expression pattern of Sema3A was compared with that of the tight-junction protein zonula occludens-1 (ZO-1), the gap-junction protein connexin43 (Cx43), or the cell proliferation marker Ki67. Immunofluorescence analysis revealed that Sema3A was present predominantly in the membrane of basal and wing cells of the intact corneal epithelium. The expression of Sema3A at the basal side of basal cells was increased in the peripheral epithelium compared with that in the central region. Sema3A was detected in all layers at the leading edge of the migrating corneal epithelium at 6 h after central epithelial debridement. The expression of Sema3A was markedly up-regulated in the basal and lateral membranes of columnar basal cells apparent in the thickened, newly healed epithelium at 1 day after debridement, but it had largely returned to the normal pattern at 3 days after debridement. The expression of ZO-1 was restricted to superficial epithelial cells and remained mostly unchanged during the wound healing process. The expression of Cx43 in basal cells was down-regulated at the leading edge of the migrating epithelium but was stable in the remaining portion of the epithelium. Ki67 was not detected in basal cells of the central epithelium at 1 day after epithelial debridement, when Sema3A was prominently expressed. Immunoblot analysis showed that the abundance of Sema3A in the central cornea was increased 1 day after epithelial debridement, whereas that of ZO-1 or Cx43 remained largely unchanged. This increase in Sema3A expression was accompanied by up-regulation of the Sema3A coreceptor neuropilin-1. Our observations have thus shown that the expression of Sema3A is increased markedly in basal cells of the newly healed corneal epithelium, and that this up-regulation of Sema3A is not associated with cell proliferation. They further suggest that Sema3A might play a role in the regulation of corneal epithelial wound healing.     

The Detailed Localization Pattern of Na+/K+/2Cl? Cotransporter Type 2 and Its Related Ion Transport System in the Rat Endolymphatic Sac

The endolymphatic sac (ES) is a part of the membranous labyrinth. ES is believed to perform endolymph absorption, which is dependent on several ion transporters, including Na⁺/K⁺/2Cl⁻ cotransporter type 2 (NKCC-2) and Na⁺/K⁺-ATPase. NKCC-2 is typically recognized as a kidney-specific ion transporter expressed in the apical membrane of the absorptive epithelium. NKCC-2 expression has been confirmed only in the rat and human ES other than the kidney, but the detailed localization features of NKCC-2 have not been investigated in the ES. Thus, we evaluated the specific site expressing NKCC-2 by immunohistochemical assessment. NKCC-2 expression was most frequently seen in the intermediate portion of the ES, where NKCC-2 is believed to play an important role in endolymph absorption. In addition, NKCC-2 expression was also observed on the apical membranes of ES epithelial cells, and Na⁺/K⁺-ATPase coexpression was observed on the basolateral membranes of ES epithelial cells. These results suggest that NKCC-2 performs an important role in endolymph absorption and that NKCC-2 in apical membranes and Na⁺/K⁺-ATPase in basolateral membranes work coordinately in the ES in a manner similar to that in renal tubules. (J Histochem Cytochem 58:759–763, 2010)     

Mechanosensitive Ca2+ permeant cation channels in human prostate tumor cells

The acquisition of cell motility plays a critical role in the spread of prostate cancer (PC), therefore, identifying a sensitive step that regulates PC cell migration should provide a promising target to block PC metastasis. Here, we report that a mechanosensitive Ca²⁺-permeable cation channel (MscCa) is expressed in the highly migratory/invasive human PC cell line, PC-3 and that inhibition of MscCa by Gd³⁺ or GsMTx-4 blocks PC-3 cell migration and associated elevations in [Ca²⁺]_i. Genetic suppression or overexpression of specific members of the canonical transient receptor potential Ca²⁺ channel family (TRPC1 and TRPC3) also inhibit PC-3 cell migration, but they do so by mechanisms other that altering MscCa activity. Although LNCaP cells are nonmigratory, they also express relatively large MscCa currents, indicating that MscCa expression alone cannot confer motility on PC cells. MscCa in both cell lines show similar conductance and ion selectivity and both are functionally coupled via Ca²⁺ influx to a small Ca²⁺-activated K⁺ channel. However, MscCa in PC-3 and LNCaP cell patches show markedly different gating dynamics—while PC-3 cells typically express a sustained, non-inactivating MscCa current, LNCaP cells express a mechanically-fragile, rapidly inactivating MscCa current. Moreover, mechanical forces applied to the patch, can induce an irreversible transition from the transient to the sustained MscCa gating mode. Given that cancer cells experience increasing compressive and shear forces within a growing tumor, a similar shift in channel gating in situ would have significant effects on Ca²⁺ signaling that may play a role in tumor progression.     

Nectin-3 expression is elevated in limbal epithelial side population cells with strongly expressed stem cell markers

Corneal epithelial stem cells (CESCs) are essential for maintaining the ocular surface. However, the lack of surface markers for CESCs remains a serious obstacle in the identification of CESCs. Previously, we showed that rabbit limbal epithelial side population (rLE-SP) cells exhibited stem cell phenotypes including increased expression of CD61, a marker for mouse hematopoietic stem cells. Here, we demonstrate that nectin-3, an immunoglobulin-like cell-cell adhesion molecule, is highly expressed in rLE-SP cells. Additionally, nectin-3⁺ cells were significantly enriched among CD61⁺rLE-SP cells as compared to CD61⁻rLE-SP cells. In mouse bone marrow side population cells, a correlation between expression of nectin-3 and CD61 was also observed. These data strongly suggest that nectin-3 may contribute to the identification of CESCs.     

Histone deacetylase inhibitor- and PMA-induced upregulation of PMCA4b enhances Ca clearance from MCF-7 breast cancer cells

The expression of the plasma membrane Ca²⁺ ATPase (PMCA) isoforms is altered in several types of cancer cells suggesting that they are involved in cancer progression. In this study we induced differentiation of MCF-7 breast cancer cells by histone deacetylase inhibitors (HDACis) such as short chain fatty acids (SCFAs) or suberoylanilide hydroxamic acid (SAHA), and by phorbol 12-myristate 13-acetate (PMA) and found strong upregulation of PMCA4b protein expression in response to these treatments. Furthermore, combination of HDACis with PMA augmented cell differentiation and further enhanced PMCA4b expression both at mRNA and protein levels. Immunocytochemical analysis revealed that the upregulated protein was located mostly in the plasma membrane. To examine the functional consequences of elevated PMCA4b expression, the characteristics of intracellular Ca²⁺ signals were investigated before and after differentiation inducing treatments, and also in cells overexpressing PMCA4b. The increased PMCA4b expression – either by treatment or overexpression – led to enhanced Ca²⁺ clearance from the stimulated cells. We found pronounced PMCA4 protein expression in normal breast tissue samples highlighting the importance of this pump for the maintenance of mammary epithelial Ca²⁺ homeostasis. These results suggest that modulation of Ca²⁺ signaling by enhanced PMCA4b expression may contribute to normal development of breast epithelium and may be lost in cancer.     

The anti-anginal drug fendiline elevates cytosolic Ca(2+) in rabbit corneal epithelial cells

The effect of the anti-anginal drug fendiline on intracellular free Ca(2+) levels ([Ca(2+)](i)) in a rabbit corneal epithelial cell line (SIRC) was explored using fura-2 as a fluorescent Ca(2+) indicator. At a concentration above 1 microM, fendiline increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 7 microM. The [Ca(2+)](i) response consisted of an immediate rise and an elevated phase. Extracellular Ca(2+) removal decreased half of the [Ca(2+)](i )signal. Fendiline induced quench of fura-2 fluorescence by Mn(2+) (50 microM), suggesting the presence of Ca(2+) influx across the plasma membrane. This Ca(2+) influx was abolished by La(3+) (50 microM), but was insensitive to dihydropyridines, verapamil and diltiazem. Fendiline (10 microM)-induced store Ca(2+) release was largely reduced by pretreatment with thapsigargin (1 microM) (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+). Conversely, pretreatment with 10 microM fendiline abolished thapsigargin-induced Ca(2+) release. Fendiline (10 microM)-induced Ca(2+) release was not altered by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Cumulatively, this study shows that fendiline induced concentration-dependent [Ca(2+)](i )increases in corneal epithelial cells by releasing the endoplasmic reticulum Ca(2+) in a phospholipase C-independent manner, and by causing Ca(2+) influx.     

Up-regulation of ZO-1 expression and barrier function in cultured human corneal epithelial cells by substance P

The effects of the sensory neurotransmitter substance P on the expression of tight junction proteins and on barrier function in human corneal epithelial cells were investigated. The expression of ZO-1, but not that of occludin or claudin-1, was increased by substance P in a concentration- and time-dependent manner. This effect was inhibited by the NK-1 receptor antagonist GR82334 and by KN62, an inhibitor of Ca²⁺- and calmodulin-dependent protein kinase II. Substance P also increased the transepithelial electrical resistance of a cell monolayer in a manner sensitive to GR82334. Substance P may therefore play a role in maintenance of tight junctions in the corneal epithelium.     

The potassium ion channel opener NS1619 inhibits proliferation and induces apoptosis in A2780 ovarian cancer cells

Diverse types of voltage-gated potassium (K⁺) channels have been shown to be involved in regulation of cell proliferation. The maxi-conductance Ca²⁺-activated K⁺ channels (BK channels) may play an important role in the progression of human cancer. To explore the role of BK channels in regulation of apoptosis in human ovarian cancer cells, the effects of the specific BK channel activator NS1619 on induction of apoptosis in A2780 cells were observed. Following treatment with NS1619, cell proliferation was measured by MTT assay. Apoptosis of A2780 cells pretreated with NS1619 was detected by agarose gel electrophoresis of cellular DNA and flow cytometry. Our data demonstrate that NS1619 inhibits the proliferation of A2780 cells in a dosage and time dependent manner IC₅₀ = 31.1 μM, for 48 h pretreatment and induces apoptosis. Western blot analyses showed that the anti-proliferation effect of NS1619 was associated with increased expression of p53, p21, and Bax. These results indicate that BK channels play an important role in regulating proliferation of human ovarian cancer cells and may induce apoptosis through induction of p21^Cip1 expression in a p53-dependent manner.     

The bestrophin- and TMEM16A-associated Ca2+-activated Cl– channels in vascular smooth muscles

The presence of Ca²⁺-activated Cl^– currents (I_Cl(Ca)) in vascular smooth muscle cells (VSMCs) is well established. I_Cl(Ca) are supposedly important for arterial contraction by linking changes in [Ca²⁺]_i and membrane depolarization. Bestrophins and some members of the TMEM16 protein family were recently associated with I_Cl(Ca). Two distinct I_Cl(Ca) are characterized in VSMCs; the cGMP-dependent I_Cl(Ca) dependent upon bestrophin expression and the ‘classical’ Ca²⁺-activated Cl^– current, which is bestrophin-independent. Interestingly, TMEM16A is essential for both the cGMP-dependent and the classical I_Cl(Ca). Furthermore, TMEM16A has a role in arterial contraction while bestrophins do not. TMEM16A’s role in the contractile response cannot be explained however only by a simple suppression of the depolarization by Cl^– channels. It is suggested that TMEM16A expression modulates voltage-gated Ca²⁺ influx in a voltage-independent manner and recent studies also demonstrate a complex role of TMEM16A in modulating other membrane proteins.     

Expression and Function of Epithelial Anoctamins

The calcium-activated chloride channel anoctamin1 (ANO1; TMEM16A) is fundamental for the function of epithelial organs. Mice lacking ANO1 expression exhibit transport defects and a pathology similar to cystic fibrosis. They also show a general defect of epithelial electrolyte transport. Here we analyzed expression of all ten members (ANO1–ANO10) in a broad range of murine tissues and detected predominant expression of ANO1, 6, 7, 8, 9, 10 in epithelial tissues, while ANO2, 3, 4, 5 are common in neuronal and muscle tissues. When expressed in Fisher Rat Thyroid (FTR) cells, all ANO proteins localized to the plasma membrane but only ANO1, 2, 6, and 7 produced Ca²⁺-activated Cl⁻ conductance, as analyzed by ATP-induced iodide quenching of YFP fluorescence. In contrast ANO9 and ANO10 suppressed baseline Cl⁻ conductance and coexpression of ANO9 with ANO1 inhibited ANO1 activity. Patch clamping of ANO-expressing FRT cells indicated that apart from ANO1 also ANO6 and 10 produced chloride currents, albeit with very different Ca²⁺ sensitivity and activation time. We conclude that each tissue expresses a set of anoctamins that form cell- and tissue-specific Ca²⁺-dependent Cl⁻ channels.     

Mechanosensitive Ca2+ permeant cation channels in human prostate tumor cells

The acquisition of cell motility plays a critical role in the spread of prostate cancer (PC), therefore, identifying a sensitive step that regulates PC cell migration should provide a promising target to block PC metastasis. Here, we report that a mechanosensitive Ca²⁺-permeable cation channel (MscCa) is expressed in the highly migratory/invasive human PC cell line, PC-3 and that inhibition of MscCa by Gd³⁺ or GsMTx-4 blocks PC-3 cell migration and associated elevations in [Ca²⁺]_i. Genetic suppression or overexpression of specific members of the canonical transient receptor potential Ca²⁺ channel family (TRPC1 and TRPC3) also inhibit PC-3 cell migration, but they do so by mechanisms other that altering MscCa activity. Although LNCaP cells are nonmigratory, they also express relatively large MscCa currents, indicating that MscCa expression alone cannot confer motility on PC cells. MscCa in both cell lines show similar conductance and ion selectivity and both are functionally coupled via Ca²⁺ influx to a small Ca²⁺-activated K⁺ channel. However, MscCa in PC-3 and LNCaP cell patches show markedly different gating dynamics—while PC-3 cells typically express a sustained, non-inactivating MscCa current, LNCaP cells express a mechanically-fragile, rapidly inactivating MscCa current. Moreover, mechanical forces applied to the patch, can induce an irreversible transition from the transient to the sustained MscCa gating mode. Given that cancer cells experience increasing compressive and shear forces within a growing tumor, a similar shift in channel gating in situ would have significant effects on Ca²⁺ signaling that may play a role in tumor progression.     

Generation and Analysis of Serine Protease Inhibitor Kazal Type 3-Cre Driver Mice

Serine protease inhibitor Kazal type 1 (SPINK1; mouse homologue Spink3) was initially discovered as a trypsin-specific inhibitor in the pancreas. However, previous studies have suggested that SPINK1/Spink3 is expressed in a wide range of normal tissues and tumors, although precise characterization of its gene expression has not been described in adulthood. To further analyze Spink3 expression, we generated two mouse lines in which either lacZ or Cre recombinase genes were inserted into the Spink3 locus by Cre-loxP technology. In Spink3^lacZ mice, β-galactosidase activity was found in acinar cells of the pancreas and kidney, as well as epithelial cells of the bronchus in the lung, but not in the gastrointestinal tract or liver. Spink3^cre knock-in mice were crossed with Rosa26 reporter (R26R) mice to monitor Spink3 promoter activity. In Spink3^cre;R26R mice, β-galactosidase activity was found in acinar cells of the pancreas, kidney, lung, and a small proportion of cells in the gastrointestinal tract and liver. These data suggest that Spink3 is widely expressed in endoderm-derived tissues, and that Spink3^cre knock-in mice are a useful tool for establishment of a conditional knockout mice to analyze Spink3 function not only in normal tissues, but also in tumors that express SPINK1/Spink3.     

Interactions of extracellular collagen and corneal fibroblasts: Morphologic and biochemical changes of rabbit corneal cells cultured in a collagen matrix

Summary Corneal fibroblasts, also known as keratocytes are surrounded by an extracellular matrix of collagen in vivo. To understand the physiology and pathology of these corneal fibroblasts, it is important to study their interactions with this extracellular matrix. We cultured rabbit corneal fibroblasts on tissue culture plastic dishes or in a hydrated collagen gel and compared the changes in morphology and mitotic activity. Corneal fibroblasts on plastic dishes were flattened and widely spread, whereas those in collagen gel became spindle-shaped with long processes. Examination with an electron microscope revealed that the corneal fibroblasts in collagen gel formed gap junctions with neighboring cells. Gap junctions were hardly ever observed between corneal fibroblasts cultured on plastic dishes. Corneal fibroblasts cultured in a collagen matrix showed much less incorporation of [³H]thymidine than did corneal fibroblasts cultured on plastic, and this incorporation decreased with increasing concentration of collagen. Our present results suggest that the morphologic and biochemical characteristics of corneal fibroblasts cultured in collagen gel are different from those cultured on plastic. This research was supported in part by grants from the Ministry of Education, Science and Culture of Japan, by a grant from Osaka Eye Bank, Osaka, Japan, and by an intramural research fund of Kinki University. Part of this research was presented at the annual meeting of the Japanese Ophthalmological Society (May 1985) at Kyoto, Japan, and at the annual meeting of the Association for Research in Vision and Ophthalmology (May 1987) at Sarasota, FL.     

Calcium-dependent facilitation and graded deactivation of store-operated calcium entry in fetal skeletal muscle

Activation of store-operated Ca²⁺ entry (SOCE) into the cytoplasm requires retrograde signaling from the intracellular Ca²⁺ release machinery, a process that involves an intimate interaction between protein components on the intracellular and cell surface membranes. The cellular machinery that governs the Ca²⁺ movement in muscle cells is developmentally regulated, reflecting maturation of the junctional membrane structure as well as coordinated expression of related Ca²⁺ signaling molecules. Here we demonstrate the existence of SOCE in freshly isolated skeletal muscle cells obtained from embryonic days 15 and 16 of the mouse embryo, a critical stage of muscle development. SOCE in the fetal muscle deactivates incrementally with the uptake of Ca²⁺ into the sarcoplasmic reticulum (SR). A novel Ca²⁺-dependent facilitation of SOCE is observed in cells transiently exposed to high cytosolic Ca²⁺. Our data suggest that cytosolic Ca²⁺ can facilitate SOCE whereas SR luminal Ca²⁺ can deactivate SOCE in the fetal skeletal muscle. This cooperative mechanism of SOCE regulation by Ca²⁺ ions not only enables tight control of SOCE by the SR membrane, but also provides an efficient mechanism of extracellular Ca²⁺ entry in response to physiological demand. Such Ca²⁺ signaling mechanism would likely contribute to contraction and development of the fetal skeletal muscle.