Integrin-dependent actomyosin contraction regulates epithelial cell scattering

The scattering of Madin-Darby canine kidney cells in vitro mimics key aspects of epithelial-mesenchymal transitions during development, carcinoma cell invasion, and metastasis. Scattering is induced by hepatocyte growth factor (HGF) and is thought to involve disruption of cadherin-dependent cell-cell junctions. Scattering is enhanced on collagen and fibronectin, as compared with laminin1, suggesting possible cross talk between integrins and cell-cell junctions. We show that HGF does not trigger any detectable decrease in E-cadherin function, but increases integrin-mediated adhesion. Time-lapse imaging suggests that tension on cell-cell junctions may disrupt cell-cell adhesion. Varying the density and type of extracellular matrix proteins shows that scattering correlates with stronger integrin adhesion and increased phosphorylation of the myosin regulatory light chain. To directly test the role of integrin-dependent traction forces, substrate compliance was varied. Rigid substrates that produce high traction forces promoted scattering, in comparison to more compliant substrates. We conclude that integrin-dependent actomyosin traction force mediates the disruption of cell-cell adhesion during epithelial cell scattering.     

Calcium regulates independently ciliary beat and cell contraction in Paramecium cells

Intracellular Ca(2+) concentration is a well-known signal regulator for various physiological activities. In many cases, Ca(2+) simultaneously regulates individual functions in single cells. How can Ca(2+) regulate these functions independently? In Paramecium cells, the contractile cytoskeletal network and cilia are located close to each other near the cell surface. Cell body contraction, ciliary reversal, and rises in ciliary beat frequency are regulated by intracellular Ca(2+) concentration. However, they are not always triggered simultaneously. We injected caged calcium into Paramecium caudatum cells and continuously applied weak ultraviolet light to the cells to slowly increase intracellular Ca(2+) concentration. The cell bodies began to contract just after the start of ultraviolet light application, and the degree of contraction increased gradually thereafter. On the other hand, cilia began to reverse 1.4s after the start of ultraviolet application and reversed completely within 100ms. Ciliary beat frequency in the reverse direction was significantly higher than in the normal direction. These results indicate that cell body contraction is regulated by Ca(2+) in a dose-dependent manner in living P. caudatum. On the other hand, ciliary reversal and rise in ciliary beat frequency are triggered by Ca(2+) in an all-or-none manner.     

Calcium regulation of normal human mammary epithelial cell growth in culture

Summary The concentration of Ca⁺⁺ in culture media profoundly affected the growth and differentiation properties of normal human mammary epithelial cells in short-term culture. In media where Ca⁺⁺ was above 0.06 mM, longevity was limited to an average of three to four cell divisions. The extended growth fraction (those cells able, to divide more than once) was only approximately 50% and diminished to zero quickly with time. Stationary cells inhibited from dividing appeared differentiated in the formation of lipid vacuoles and accumulation of α-lactalbumin. Growth of stationary cultures could be reinstituted in about half the cells, either by disruption and transfer or by a reduction in Ca⁺⁺ to less than 0.08 mM. The reduction of Ca⁺⁺ to levels below 0.08 mM extended the longevity of normal cells to eight to nine divisions. The extended growth fraction was 100%. Under these conditions, cells did not differentiate. The effects of Ca⁺⁺ on growth and differentiation were specific (Mg⁺⁺ and Mn⁺⁺ variations were without effect) and reversible and in many respects resembled Ca⁺⁺ effects on epidermal cells. One major difference is that the dual pathways of growth and differentiation in mammary cells were controlled by glucocorticoid and insulin. Based on the kinetics of the reversible Ca⁺⁺-induced coupling and uncoupling of proliferation and the program of differentiation, we propose that Ca⁺⁺ may be an essential trigger for cell divisions that commit a mammary cell to differentiate progressively in a permissive hormonal milieu. This study was supported by grants NIH-CA18175 and CA36399 and an institutional grant from the United Foundation of Greater Detroit.     

Calcium dependence and contraction in somite formation

The existence of a calcium-dependent contractile process in the formation of somites from segmental plate mesoderm was investigated using a Ca2+ agonist and Ca2+ and calmodulin antagonists. The contribution of cell movement and apical constriction in the segmentation process were assessed using SEM of normal and drug-treated somite and segmental plate tissue. Explants that contained segmental plates of stage 14-15 chick embryos were cultured on vitelline membranes in calcium- and magnesium-free (CMF) Hands' solution, liquid culture medium, and medium containing drugs. Ca2+ ionophore A23187 promoted the rapid completion of one new somite pair. CMF halted segmentation. The Ca2+ antagonists verapamil and papaverine reversibly inhibited segmentation. Theophylline did not inhibit segmentation, suggesting that the effects of the Ca2+ antagonists are not due to inhibition of phosphodiesterase activity. These results suggest that somitogenesis is Ca2+-dependent. Two drugs that inhibit the binding of calmodulin, chlorpromazine and trifluoperazine (TFP), halted segmentation. The inhibitory effect of TFP was reversible. The effects of TFP on somites were compared with those of cytochalasin D. The contribution of microtubules to cell shape and movement in somitogenesis was examined by incubation with nocodazole, a reversible inhibitor of tubulin polymerization. Cell elongation and somitogenesis were inhibited.     

Calcium transport systems in the LLC-PK1 renal epithelial established cell line

ATP-dependent calcium uptake was measured in membrane vesicles prepared from the renal epithelial LLC-PK1 established cell line. The relative contribution of the nonmitochondrial versus the mitochondrial calcium uptake is larger in LLC-PK1 cell homogenates than in homogenates from renal cortex. Two types of calcium pump, characterized by the formation of calcium-dependent phosphointermediates of 135 kDa and 115 kDa, were found in membrane fractions from LLC-PK1 cells. The 135 kDa calcium pump was also detected by 125I-labelled calmodulin overlay. Although the subcellular localization in LLC-PK1 cell membranes could not be unambiguously determined, it is conceivable that the 135 kDa and the 115 kDa molecules represent the plasma membrane calcium pump and the endoplasmic reticulum calcium pump respectively, in agreement with what was found for renal cortex preparations. Extravesicular sodium partially inhibits ATP-driven calcium uptake in a plasma-membrane-enriched fraction of the LLC-PK1 cells. The effect is potentiated by a vesicle inside-negative membrane potential. Although the effect is less pronounced than in renal cortex basal-lateral membranes, this observation suggests that an Na+-Ca2+ exchange mechanism is also present in LLC-PK1 cells. ATP-dependent calcium uptake in nonmitochondrial intracellular stores was investigated, using saponin-permeabilized cells. Permeabilized LLC-PK1 cells lowered the free calcium concentration in the medium to less than 0.4 microM. More than 60% of the accumulated calcium can be released by addition of inositol 1,4,5-trisphosphate. Our data indicate that the LLC-PK1 cell line can be successfully used as model system for the study of renal calcium handling.     

Calcium regulation of muscle contraction.

Calcium triggers contraction by reaction with regulatory proteins that in the absence of calcium prevent interaction of actin and myosin. Two different regulatory systems are found in different muscles. In actin-linked regulation troponin and tropomyosin regulate actin by blocking sites on actin required for complex formation with myosin; in myosin-linked regulation sites on myosin are blocked in the absence of calcium. The major features of actin control are as follows: there is a requirement for tropomyosin and for a troponin complex having three different subunits with different functions; the actin displays a cooperative behavior; and a movement of tropomyosin occurs controlled by the calcium binding on troponin. Myosin regulation is controlled by a regulatory subunit that can be dissociated in scallop myosin reversibly by removing divalent cations with EDTA. Myosin control can function with pure actin in the absence of tropomyosin. Calcium binding and regulation of molluscan myosins depend on the presence of regulatory light chains. It is proposed that the light chains function by sterically blocking myosin sites in the absence of calcium, and that the "off" state of myosin requires cooperation between the two myosin heads. Both myosin control and actin control are widely distributed in different organisms. Many invertebrates have muscles with both types of regulation. Actin control is absent in the muscles of molluscs and in several minor phyla that lack troponin. Myosin control is not found in striated vertebrate muscles and in the fast muscles of crustacean decapods, although regulatory light chains are present. While in vivo myosin control may not be excluded from vertebrate striated muscles, myosin control may be absent as a result of mutations of the myosin heavy chain.     

Calcium homeostasis of epithelial cells

1. Cytosolic free Ca2+ is an important regulator of ion transport processes in epithelial cells. 2. Free Ca2+ concentration is regulated by a concerted action of Ca2+ transport systems in plasma membranes and intracellular organelles. 3. These transport systems were studied in intestinal and renal cortical cells with emphasis on the transport capacities and Ca2+ affinities. 4. Ca2+ accumulation by permeabilized cells was compared to Ca2+ uptake by isolated organelles and membrane fractions. 5. Effects induced by cell or organelle isolation methods and the influence of temperature and pH on Ca2+ transport capacities were studied.     

Calcium mobilization and muscle contraction induced by acetylcholine in swine trachealis

The roles of Ca²⁺ mobilization in development of tension induced by acetylcholine (ACh, 0.1–100 µM) in swine tracheal smooth muscle strips were studied. Under control conditions, ACh induced a transient increase in free cytosolic calcium concentration ([Ca²⁺]_i) that declined to a steady-state level. The peak increase in [Ca²⁺]_i correlated with the magnitude of tension at each [ACh] after a single exposure to ACh, while the steady-state [Ca²⁺]_i did not. Removal of extracellular Ca²⁺ had little effect on peak [Ca²⁺]_i but greatly reduced steady-state increases in [Ca²⁺]_i and tension. Verapamil inhibited steady-state [Ca²⁺]_i only at [ACh]<1 µM. After depletion of internal Ca²⁺ stores by 10 min exposure to ACh in Ca²⁺-free solution and then washout of ACh for 5 min in Ca²⁺-free solution, simultaneous re-exposure to ACh in the presence of 2.5 mM Ca²⁺ increased [Ca²⁺]_i to the control steady-state level without overshoot. The tension attained was the same as control for each [ACh] used. Continuous exposure to successively increasing [ACh] (0.1–100 µM) also reduced the overshoot of [Ca²⁺]_i at 10 and 100 µM ACh, yet tension reached control levels at each [ACh] used. We conclude that the steady-state increase in [Ca²⁺]_i is necessary for tension maintenance and is dependent on Ca²⁺ influx through voltage-gated calcium channels at 0.1 µM ACh and through a verapamil-insensitive pathway at 10 and 100 µM. The initial transient increase in calcium arises from intracellular stores and is correlated with the magnitude of tension only in muscles that have completely recovered from previous exposure to agonists.     

Lipid metabolism and Calcium signaling in epithelial ovarian cancer

Epithelial Ovarian cancer (EOC) is the deadliest gynecologic malignancy and represents the fifth leading cause of all cancer-related deaths in women. The majority of patients are diagnosed at an advanced stage of the disease that has spread beyond the ovaries to the peritoneum or to distant organs (stage FIGO III-IV) with a 5-year overall survival of about 29%. Consequently, it is necessary to understand the pathogenesis of this disease. Among the factors that contribute to cancer development, lipids and ion channels have been described to be associated to cancerous diseases particularly in breast, colorectal and prostate cancers. Here, we reviewed the literature data to determine how lipids or lipid metabolites may influence EOC risk or progression. We also highlighted the role and the expression of the calcium (Ca²⁺) and calcium-activated potassium (KCa) channels in EOC and how lipids might regulate them. Although lipids and some subclasses of nutritional lipids may be associated to EOC risk, lipid metabolism of LPA (lysophosphatidic acid) and AA (arachidonic acid) emerges as an important signaling network in EOC. Clinical data showed that they are found at high concentrations in EOC patients and in vitro and in vivo studies referred to them as triggers of the Ca²⁺entry in the cancer cells inducing their proliferation, migration or drug resistance. The cross-talk between lipid mediators and Ca²⁺ and/or KCa channels needs to be elucidated in EOC in order to facilitate the understanding of its outcomes and potentially suggest novel therapeutic strategies including treatment and prevention.     

Thrombin-induced contraction in alveolar epithelial cells probed by traction microscopy.

Contractile tension of alveolar epithelial cells plays a major role in the force balance that regulates the structural integrity of the alveolar barrier. The aim of this work was to study thrombin-induced contractile forces of alveolar epithelial cells. A549 alveolar epithelial cells were challenged with thrombin, and time course of contractile forces was measured by traction microscopy. The cells exhibited basal contraction with total force magnitude 55.0 +/- 12.0 nN (mean +/- SE, n = 12). Traction forces were exerted predominantly at the cell periphery and pointed to the cell center. Thrombin (1 U/ml) induced a fast and sustained 2.5-fold increase in traction forces, which maintained peripheral and centripetal distribution. Actin fluorescent staining revealed F-actin polymerization and enhancement of peripheral actin rim. Disruption of actin cytoskeleton with cytochalasin D (5 microM, 30 min) and inhibition of myosin light chain kinase with ML-7 (10 microM, 30 min) and Rho kinase with Y-27632 (10 microM, 30 min) markedly depressed basal contractile tone and abolished thrombin-induced cell contraction. Therefore, the contractile response of alveolar epithelial cells to the inflammatory agonist thrombin was mediated by actin cytoskeleton remodeling and actomyosin activation through myosin light chain kinase and Rho kinase signaling pathways. Thrombin-induced contractile tension might further impair alveolar epithelial barrier integrity in the injured lung.     

Calcium deficiency-induced and TRP channel-regulated IGF1R-PI3K-Akt signaling regulates abnormal epithelial cell proliferation

Calcium deficiency causes abnormal colonic growth and increases colon cancer risk with poorly understood mechanisms. Here we elucidate a novel signaling mechanism underlying the Ca²⁺ deficiency-induced epithelial proliferation using a unique animal model. The zebrafish larval yolk sac skin contains a group of Ca²⁺-transporting epithelial cells known as ionocytes. Their number and density increases dramatically when acclimated to low [Ca²⁺] environments. BrdU pulse-labeling experiments suggest that low [Ca²⁺] stimulates pre-existing ionocytes to re-enter the cell cycle. Low [Ca²⁺] treatment results in a robust and sustained activation of IGF1R-PI3K-Akt signaling in these cells exclusively. These ionocytes specifically express Igfbp5a, a high-affinity and specific binding protein for insulin-like growth factors (IGFs) and the Ca²⁺-selective channel Trpv5/6. Inhibition or knockdown of Igfbp5a, IGF1 receptor, PI3K, and Akt attenuates low [Ca²⁺]-induced ionocyte proliferation. The role of Trpv5/6 was investigated using a genetic mutant, targeted knockdown, and pharmacological inhibition. Loss-of-Trpv5/6 function or expression results in elevated pAkt levels and increased ionocyte proliferation under normal [Ca²⁺]. These increases are eliminated in the presence of an IGF1R inhibitor, suggesting that Trpv5/6 represses IGF1R-PI3K-Akt signaling under normal [Ca²⁺]. Intriguingly, blockade of Trpv5/6 activity inhibits the low [Ca²⁺]-induced activation of Akt. Mechanistic analyses reveal that the low [Ca²⁺]-induced IGF signaling is mediated through Trpv5/6-associated membrane depolarization. Low extracellular [Ca²⁺] results in a similar amplification of IGF-induced PI3K-PDK1-Akt signaling in human colon cancer cells in a TRPV6-dependent manner. These results uncover a novel and evolutionarily conserved signaling mechanism that contributes to the abnormal epithelial proliferation associated with Ca²⁺ deficiency.     

Reactivation of intestinal epithelial cell brush border motility: ATP- dependent contraction via a terminal web contractile ring

Various models have been put forward suggesting ways in which brush borders from intestinal epithelial cells may be motile. Experiments documenting putative brush border motility have been performed on isolated brush borders and have generated models suggesting microvillar retraction or microvillar rootlet interactions. The reported Ca++ ATP- induced retraction of microvilli has been shown, instead, to be microvillar dissolution in response to Ca++ and not active brush border motility. I report here studies on the reactivation of motility in intact sheets of isolated intestinal epithelium. Whole epithelial sheets were glycerinated, which leaves the brush border and intercellular junctions intact, and then treated with ATP, PPi, ITP, ADP, GTP, or delta S-ATP. Analysis by video enhanced differential interference-contrast microscopy and thin-section transmission electron microscopy reveals contractions in the terminal web region causing microvilli to be fanned apart in response to ATP and delta S-ATP but not in response to ADP, PPi, ITP, or GTP. Electron microscopy reveals that the contractions occur at the level of the intermediate junction in a circumferential constriction which can pull cells completely apart. This constriction occurs in a location occupied by an actin- containing circumferential band of filaments, as demonstrated by S-1 binding, which completely encircles the terminal web at the level of the intermediate junction. Upon contraction, this band becomes denser and thicker. Since myosin, alpha-actinin and tropomyosin, in addition to actin, have been localized to this region of the terminal web, it is proposed that the intestinal epithelial cell can be motile via a circumferential terminal web contractile ring analogous to the contractile ring of dividing cells.     

Calcium oscillations in gingival epithelial cells infected with Porphyromonas gingivalis

The periodontal pathogen Porphyromonas gingivalis modulates epithelial cell signal transduction pathways including Ca2+ signaling, and internalizes within the host cell cytoplasm. Since nuclear and cytoplasmic [Ca2+] increases can induce different host cell responses, P. gingivalis-related [Ca2+] changes in these compartments were measured by digital fluorescent imaging microscopy. Non-deconvolved and deconvolved fura-2 images showed that P. gingivalis exposure caused human gingival epithelial cells cultured in physiologic [Ca2+] levels to undergo sustained oscillations of [Ca2+] in nuclear and cytoplasmic spaces. However, P. gingivalis invasion was not tightly correlated with intracellular [Ca2+] oscillations, since invasion could significantly precede, or even occur in the absence of, oscillations. [Ca2+] oscillations required a Ca2+ influx, which was completely inhibited by La3+ or 2-APB (2-aminoethoxydiphenyl borate), indicating Ca2+ entry was via a Ca(2+)-permeable channel. Ca2+ entry was likely not via a store-operated channel, since Ca2+ release from intracellular stores was not observed during cellular uptake of P. gingivalis. Hence, uptake of P. gingivalis in gingival epithelial cells induces oscillations in nuclear and cytoplasmic spaces by activating a Ca2+ influx through Ca2+ channels.     

Calcium stimulates ornithine decarboxylase activity in cultured mammalian epithelial cells

Ornithine decarboxylase (ODC) activity usually rises to a peak a few hours after a trophic stimulus. The stimulation of ODC has been shown to depend on extracellular calcium in several in vitro eukaryotic systems. We have investigated the effect of calcium concentration on ODC activity and have found that ODC is stimulated when CaCl2 alone is added to calcium-deprived cells. Epithelial cells from calf esophagus were cultured and grown until stratified. Replacement of medium with fresh serum-free medium resulted in stimulation of ODC activity, which peaked at 4 hours and declined to basal level by 10 hours. Subsequent depletion of Ca2+ either by addition of ethylene glycol bis (beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) or by replacement of medium with Ca2+-free medium, resulted in obliteration of ODC activity 4 hours later. Conversely, cultures in which medium was replaced with Ca2+-free medium and at 10 hours were repleted with Ca2+ (either by addition of CaCl2 or by replacement of medium with Ca2+-containing medium) exhibited a pronounced elevation of ODC activity 4 hours later. ODC activity peaked at 6 hours after the addition of CaCl2 and declined by 8 hours. The effect was elicited by a wide range of concentrations of added Ca2+ from 0.1 mM to 4.0 mM, but was maximal at 1.0 mM. ODC activity was totally abolished if either cycloheximide (10 micrograms/ml) or putrescine (10 mM) was added to cultures immediately prior to Ca2+ addition. Actinomycin D (2, 5, or 10 micrograms/ml) added 30 minutes before Ca2+ did not prevent the stimulation of ODC by added Ca2+. Stimulation by Ca2+ is dependent on (1) absence of Ca2+ during the initial 10-hour incubation and (2) duration of incubation in Ca2+-free medium prior to Ca2+ replenishment. The results indicate that Ca2+ can increase ODC in epithelial cells exposed to Ca2+-depleted medium and that the increase in ODC depends on protein synthesis but is not inhibited by actinomycin D.     

Calcium transport in epithelial cells of the intestine and kidney

The central role of 1α,25-dihydroxyvitamin D₃ in the regulation of calcium balance is well established. By increasing the absorption of calcium in the intestine and the reabsorption of filtered calcium in the kidney tubule, the hormone maintains an appropriate calcium balance. The cellular mechanisms that underlie the increase in calcium transport in epithelial cells in response to 1α,25-dihydroxyvitamin D₃ are beginning to be defined. These events include an increase in the movement of calcium across the apical membrane of the cell, an increase in the movement of calcium across the cell, and an increase in the extrusion of calcium at the basolateral portion of the cell. In this Prospects article, I will discuss the nature of the various processes and proteins involved in transcellular calcium movement, and I will attempt to highlight various future areas of research.