Structurally diverse peroxisome proliferator-activated receptor agonists induce apoptosis in human uro-epithelial cells by a receptor-independent mechanism involving store-operated calcium channels

Objectives:  Peroxisome proliferator-activated receptors (PPARs) are implicated in epithelial cell proliferation and differentiation, but investigation has been confounded by potential off-target effects of some synthetic PPAR ligands. Our aim was to determine mechanisms underlying the pro-apoptotic effect of synthetic PPAR agonists in normal human bladder uro-epithelial (urothelial) cells and to reconcile this with the role of PPARs in urothelial cytodifferentiation.
Materials and methods:  Normal human urothelial (NHU) cells were grown as non-immortal lines in vitro and exposed to structurally diverse agonists ciglitazone, troglitazone, rosiglitazone (PPARγ), ragaglitazar (PPARα/γ), fenofibrate (PPARα) and L165041 (PPARβ/δ).
Results:  NHU cells underwent apoptosis following acute exposure to ciglitazone, troglitazone or ragaglitazar, but not fenofibrate, L165041 or rosiglitazone, and this was independent of ERK or p38 MAP-kinase activation. Pro-apoptotic agonists induced sustained increases in intracellular calcium, whereas removal of extracellular calcium altered the kinetics of ciglitazone-mediated calcium release from sustained to transient. Cell death was accompanied by plasma-membrane disruption, loss of mitochondrial membrane-potential and caspase-9/caspase-3 activation. PPARγ-mediated apoptosis was unaffected following pre-treatment with PPARγ antagonist T0070907 and was strongly attenuated by store-operated calcium channel (SOC) inhibitors 2-APB and SKF-96365.
Conclusions:  Our results provide a mechanistic basis for the ability of some PPAR agonists to induce death in NHU cells and demonstrate that apoptosis is mediated via PPAR-independent mechanisms, involving intracellular calcium changes, activation of SOCs and induction of the mitochondrial apoptotic pathway.     

Coordinated waves of actomyosin flow and apical cell constriction immediately after wounding

Epithelial wound healing relies on tissue movements and cell shape changes. Our work shows that, immediately after wounding, there was a dramatic cytoskeleton remodeling consisting of a pulse of actomyosin filaments that assembled in cells around the wound edge and flowed from cell to cell toward the margin of the wound. We show that this actomyosin flow was regulated by Diaphanous and ROCK and that it elicited a wave of apical cell constriction that culminated in the formation of the leading edge actomyosin cable, a structure that is essential for wound closure. Calcium signaling played an important role in this process, as its intracellular concentration increased dramatically immediately after wounding, and down-regulation of transient receptor potential channel M, a stress-activated calcium channel, also impaired the actomyosin flow. Lowering the activity of Gelsolin, a known calcium-activated actin filament–severing protein, also impaired the wound response, indicating that cleaving the existing actin filament network is an important part of the cytoskeleton remodeling process.     

Relative roles of calcium derived from intra- and extracellular sources in dynamic luteinizing hormone release from perifused pituitary cells

GnRH releases LH from pituitary gonadotropes by a calcium-dependent mechanism. Previous studies in static cell cultures have not revealed a role for intracellular-derived calcium during GnRH-stimulated LH release. In the present study we have reexamined this possibility using a perifusion system, which permits a more dynamic assessment of early cellular events. Chelation of extracellular calcium by EGTA and calcium channel blockade by methoxyverapamil prevented sustained LH release. A component of early LH release occurred independently of extracellular calcium mobilization. This previously unrecognized aspect of LH release was shown to be dependent upon intracellular calcium. The molecular mechanism by which this calcium-dependent signal is translated into a cellular response does not appear to be mediated by calmodulin or protein kinase C, whereas sustained LH release appears mediated by calmodulin. While calcium derived from extracellular sources is still viewed as the major messenger for sustained LH release, these experiments provide evidence for the involvement of intracellular-derived calcium during early GnRH-stimulated LH release.     

The role of purinergic signaling on deformation induced injury and repair responses of alveolar epithelial cells

Cell wounding is an important driver of the innate immune response of ventilator-injured lungs. We had previously shown that the majority of wounded alveolus resident cells repair and survive deformation induced insults. This is important insofar as wounded and repaired cells may contribute to injurious deformation responses commonly referred to as biotrauma. The central hypothesis of this communication states that extracellular adenosine-5' triphosphate (ATP) promotes the repair of wounded alveolus resident cells by a P2Y2-Receptor dependent mechanism. Using primary type 1 alveolar epithelial rat cell models subjected to micropuncture injury and/or deforming stress we show that 1) stretch causes a dose dependent increase in cell injury and ATP media concentrations; 2) enzymatic depletion of extracellular ATP reduces the probability of stretch induced wound repair; 3) enriching extracellular ATP concentrations facilitates wound repair; 4) purinergic effects on cell repair are mediated by ATP and not by one of its metabolites; and 5) ATP mediated cell salvage depends at least in part on P2Y2-R activation. While rescuing cells from wounding induced death may seem appealing, it is possible that survivors of membrane wounding become governors of a sustained pro-inflammatory state and thereby perpetuate and worsen organ function in the early stages of lung injury syndromes. Means to uncouple P2Y2-R mediated cytoprotection from P2Y2-R mediated inflammation and to test the preclinical efficacy of such an undertaking deserve to be explored.     

The alpha isoform of protein kinase C is involved in signaling the response of desmosomes to wounding in cultured epithelial cells

Initiation of reepithelialization upon wounding is still poorly understood. To enhance this understanding, we focus here on changes in the adhesive state of desmosomes of cultured Madin-Darby canine kidney cells in response to wounding of confluent cell sheets. Previous results show that desmosomal adhesion in Madin-Darby canine kidney cells changes from a calcium-dependent state to calcium independence in confluent cell sheets. We show that this change, which requires culture confluence to develop, is rapidly reversed upon wounding of confluent cell sheets. Moreover, the change to calcium dependence in wound edge cells is propagated to cells hundreds of micrometers away from the wound edge. Rapid transition from calcium independence to calcium dependence also occurs when cells are treated with phorbol esters that activate PKC. PKC inhibitors, including the conventional isoform inhibitor G?6976, cause rapid transition from calcium dependence to calcium independence, even in subconfluent cells. The cellular location of the alpha isoform of PKC correlates with the calcium dependence of desmosomes. Upon monolayer wounding, PKCalpha translocates rapidly to the cell periphery, becomes Triton X-100 insoluble, and also becomes concentrated in lamellipodia. The PKCalpha translocation upon wounding precedes both the increase in PKC activity in the membrane fraction and the reversion of desmosomes to calcium dependence. Specific depletion of PKCalpha with an antisense oligonucleotide increases the number of cells with calcium-independent desmosomes. These results show that PKCalpha participates in a novel signaling pathway that modulates desmosomal adhesion in response to wounding.     

Co-ordination between localized wound-induced Ca2+ signals and pre-wound serum signals is required for proliferation after mechanical injury

Abstract. The signals which initiate proliferation of endothelial cells after injury are important for selective blood vessel growth during wound healing or tumour growth. Upon mechanically wounding quiescent cells, a transient [Ca²⁺]_; increase was induced in cells at the wound edge. These same cells proliferated 18-24 h post wounding, as measured by bromodeoxyuridine incorporation. The localized Ca²⁺ signal was required specifically during wounding since blocking Ca²⁺ influx reduced proliferation by 40-50%. Proliferation also required serum since starvation reduced proliferation by 80%. Serum-starved cells proliferated if briefly primed with serum prior to wounding. The signals derived from serum and [Ca²⁺]j combined at least additively to induce proliferation. Therefore, serum priming followed by a single, transient Ca²⁺ signal induced by mechanical injury must occur in a temporally and spatially regulated manner for normal proliferation. Co-ordination between signalling cascades induced by growth factors and release from contact inhibition might be obligatory for localized re-endothelialization after injury.     

Expression of the ipomoelin gene from sweet potato is regulated by dephosphorylated proteins, calcium ion and ethylene

A wound‐inducible cDNA, ipomoelin (IPO) was isolated from the subtraction library of sweet potato (Ipomoea batatas cv. Tainung 57) and used as a molecular probe to investigate the transduction pathway of wounding signal within plant cells. Following mechanical wounding of the leaves of sweet potato, IPO mRNA accumulation peaked at 6 h and then continuously declined. However, IPO gene expression in the apical unwounded leaves began at 6 h after wounding and continued for a further 10 h. Besides mechanical wounding, methyl jasmonate (MeJA) was identified as a signal transducer leading to the accumulation of IPO mRNA. Treatment with salicylic acid reduced the production of IPO mRNA, further supporting the involvement of the octadecanoid pathway in the signal transduction of wounding in sweet potato. In addition, ethylene was involved in the signal pathway and induced the expression of the IPO gene. Furthermore, the application of okadaic acid, a protein phosphatase inhibitor, blocked the accumulation of IPO mRNA induced by MeJA or ethylene, indicating that activation of the IPO gene by both MeJA and ethylene was via dephosphorylated proteins. The presence of a calcium ion chelator or channel blockers also inhibited the expression of the IPO gene after wounding. However, investigation by confocal scanning microscopy further pointed out that mechanical wounding rather than the application of MeJA induced the accumulation of the calcium ion. These results may indicate that the calcium ion is also involved in the activation of IPO mRNA. In addition, wounding signals the accumulation of calcium ion first and then stimulates the biosynthesis of MeJA in sweet potato. Hence, the reaction sequence of signal transducers, including the calcium ion, MeJA and protein kinase/phosphatase, in the wounding signalling pathway of sweet potato is suggested in this report.     

Increased MAPK signaling during in vitro muscle wounding

Regeneration of skeletal muscle upon injury is a complex process, involving activation of satellite cells, followed by migration, fusion, and regeneration of damaged myofibers. Previous work concerning the role of the mitogen activated protein (MAP) kinase signaling pathways in muscle injury comes primarily from studies using chemically induced wounding. The purpose of this study was to test the hypothesis that physical injury to skeletal muscle cells in vitro activates the MAP kinase signaling pathways. We demonstrate that extracellular signal regulated kinases (ERKs) 1, 2, and p38 are rapidly and transiently activated in response to injury in C2C12 cells, and are primarily localized to cells adjacent to the wound bed. Culture medium from wounded cells is able to stimulate activation of p38 but not ERK in unwounded cells. These results suggest that both ERK and p38 are involved in the response of muscle cells to physical injury in culture, and reflect what is seen in whole tissues in vivo.     

Muscarinic receptor-induced calcium responses in astroglia

BACKGROUND: The objective of this study was to characterize and quantitate the calcium responses to cholinergic stimulation in individual primary rat cortical astrocytes and human 132 1N1 astrocytoma cells. Materials and Methods The fluorescent calcium probe Indo-1 AM and an attached cell analysis and sorting (ACAS) instrument were used to quantitate calcium responses in these cells. RESULTS: A concentration-dependent response to carbachol was seen in both cell types. However, carbachol was more potent and efficacious, and the response was more homogeneous in the cell line. The calcium response was mediated by the M3 subtype of muscarinic receptors. Experiments in the absence of extracellular calcium and with EGTA demonstrated that the initial calcium spike was due to calcium release from intracellular calcium stores, whereas the sustained elevation and oscillations were dependent on calcium influx. Protein kinase C exerts a feedback inhibition of these calcium responses, and appears to be involved in maintaining the elevated calcium concentration and oscillations. CONCLUSIONS: This study provided a detailed quantitation of the changes in intracellular calcium evoked in individual astroglial cells by activation of M3 muscarinic receptors. This will allow for the study of pharmacological and toxicological agents on this response.     

HUMAN WOUND REPAIR : II. Inflammatory Cells, Epithelial-Mesenchymal Interrelations, and Fibrogenesis

Connective tissue repair was studied in a series of skin wounds in young adult males. The tissues were examined at 3, 12, and 24 hr, and at 2, 3, 5, 7, 14, and 21 days after wounding. The neutrophilic leukocytes contain within membrane-bounded vacuoles some fibrin and serum protein from the wound; however, most of the granulocytes lyse and release their cytoplasmic contents into the extracellular space. The mononuclear cells undergo a series of morphologic alterations during which they develop a modest amount of relatively poorly developed rough endoplasmic reticulum and an extensive system of smooth-surfaced membranes prior to active phagocytosis. They could be clearly distinguished from immature fibroblasts by the differences in the development of their organelles, particularly the rough endoplasmic reticulum. The perivascular connective tissue adjacent to the wound contains cells which appear like poorly developed or immature fibroblasts. The development of these cells into mature fibroblasts can be followed during the different stages of wound repair. Intimate contact was observed between basal cells of the regenerated epidermis and monocytes in the wound below: cytoplasmic projections of the basal cells extended beneath the basement lamina to the surface of the monocytes. Such contacts were seen only on the 4th–7th day after wounding. Their possible significance is discussed.     

Stanniocalcin-1 Regulates Extracellular ATP-Induced Calcium Waves in Human Epithelial Cancer Cells by Stimulating ATP Release from Bystander Cells

Background

The epithelial cell response to stress involves the transmission of signals between contiguous cells that can be visualized as a calcium wave. In some cell types, this wave is dependent on the release of extracellular trinucleotides from injured cells. In particular, extracellular ATP has been reported to be critical for the epithelial cell response to stress and has recently been shown to be upregulated in tumors in vivo.

Methodology/Principal Findings

Here, we identify stanniocalcin-1 (STC1), a secreted pleiotrophic protein, as a critical mediator of calcium wave propagation in monolayers of pulmonary (A549) and prostate (PC3) epithelial cells. Addition of STC1 enhanced and blocking STC1 decreased the distance traveled by an extracellular ATP-dependent calcium wave. The same effects were observed when calcium was stimulated by the addition of exogenous ATP. We uncover a positive feedback loop in which STC1 promotes the release of ATP from cells in vitro and in vivo.

Conclusions/Significance

The results indicated that STC1 plays an important role in the early response to mechanical injury by epithelial cells by modulating signaling of extracellular ATP. This is the first report to describe STC1 as a modulator or purinergic receptor signaling.     

Tri-nucleotide receptors play a critical role in epithelial cell wound repair

The cornea plays a major role in the refraction of light to the retina. Therefore, the integrity and transparency of the corneal epithelium are critical to vision. Following injury, a combination of rapid signal transduction events and long-term cell migration are essential for wound closure. We have demonstrated previously that injury resulted in the release of nucleotides that induce the propagation of a Ca(2+) wave to neighboring cells. This suggests that nucleotides and their receptors are critical components of wound healing. Epidermal growth factor (EGF) and integrins also have been shown to play a role in injury. In this study, we demonstrate that pretreatment of cells with ATP and UTP inhibited the immediate wound response, while BzATP, ADP, and UDP did not affect this response. Tri-nucleotide pretreatment also reduced the EGF induced Ca(2+) response. Additionally, lower EC(50) concentrations of ATP and UTP triggered migration of cells that was enhanced further with EGF and was inhibited by the tripeptide, RGD. Results indicate that the desensitization induced by ATP and UTP was specific. While ADP and UDP cause a homologous desensitization of their own signal, they did not cause an inhibition of the wound response nor does BzATP. Neither Ca(2+) wave propagation nor cell migration occurred in response to beta,gamma-MeATP. Together these results lead us to hypothesize that corneal epithelial wound repair is mediated by both P2Y(2) and P2Y(4) receptors.     

Vesicular ATP is the predominant cause of intercellular calcium waves in astrocytes

Brain astrocytes signal to each other and neurons. They use changes in their intracellular calcium levels to trigger release of transmitters into the extracellular space. These can then activate receptors on other nearby astrocytes and trigger a propagated calcium wave that can travel several hundred micrometers over a timescale of seconds. A role for endogenous ATP in calcium wave propagation in hippocampal astrocytes has been suggested, but the mechanisms remain incompletely understood. Here we explored how calcium waves arise and directly tested whether endogenously released ATP contributes to astrocyte calcium wave propagation in hippocampal astrocytes. We find that vesicular ATP is the major, if not the sole, determinant of astrocyte calcium wave propagation over distances between approximately 100 and 250 microm, and approximately 15 s from the point of wave initiation. These actions of ATP are mediated by P2Y1 receptors. In contrast, metabotropic glutamate receptors and gap junctions do not contribute significantly to calcium wave propagation. Our data suggest that endogenous extracellular astrocytic ATP can signal over broad spatiotemporal scales.     

Heterogeneous response of microvascular endothelial cells to shear stress

We investigated changes in calcium concentration in cultured bovine aortic endothelial cells (BAECs) and rat adrenomedulary endothelial cells (RAMECs, microvascular) in response to different levels of shear stress. In BAECs, the onset of shear stress elicited a transient increase in intracellular calcium concentration that was spatially uniform, synchronous, and dose dependent. In contrast, the response of RAMECs was heterogeneous in time and space. Shear stress induced calcium waves that originated from one or several cells and propagated to neighboring cells. The number and size of the responding groups of cells did not depend on the magnitude of shear stress or the magnitude of the calcium change in the responding cells. The initiation and the propagation of calcium waves in RAMECs were significantly suppressed under conditions in which either purinergic receptors were blocked by suramin or extracellular ATP was degraded by apyrase. Exogenously applied ATP produced similarly heterogeneous responses. The number of responding cells was dependent on ATP concentration, but the magnitude of the calcium change was not. Our data suggest that shear stress stimulates RAMECs to release ATP, causing the increase in intracellular calcium concentration via purinergic receptors in cells that are heterogeneously sensitive to ATP. The propagation of the calcium signal is also mediated by ATP, and the spatial pattern suggests a locally elevated ATP concentration in the vicinity of the initially responding cells.     

Human keratinocytes respond to direct current stimulation by increasing intracellular calcium: preferential response of poorly differentiated cells

A direct current (DC) endogenous electric field (EF) is induced in the wound following skin injury. It is potentially implicated in the wound healing process by attracting cells and altering their phenotypes as indicated by the response to an EF of keratinocytes cultured as individual cells. To better define the signalization induced by a direct current electric field (DCEF) in human keratinocytes, we took advantage of an in vitro model more representative of the in vivo situation since it promotes cell-cell interactions and stratification. Human keratinocytes were grown into colonies. Their exposure to a DCEF of physiological intensity induced an increase of intracellular calcium. This variation of intracellular calcium resulted from an extracellular calcium influx and was mediated, at least in part, by the L-type voltage-gated calcium channel. The increase in intracellular calcium in response to a DCEF was however not observed in all the cells composing the colonies. The intracellular calcium increase was only detected in keratinocytes that didn't express involucrin, a marker of differentiated cells. These results indicate that DCEF is able to induce a specific calcium response in poorly differentiated keratinocytes. This study brings a new perspective for the understanding of the signaling mechanism of endogenous EF in reepithelialization, a critical process during skin wound healing.     

Involvement of phospholipase D in store-operated calcium influx in vascular smooth muscle cells

In non-excitable cells, sustained intracellular Ca2+ increase critically depends on influx of extracellular Ca2+. Such Ca2+ influx is thought to occur by a 'store-operated' mechanism, i.e. the signal for Ca2+ entry is believed to result from the initial release of Ca2+ from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Here we show that the depletion of cellular Ca2+ stores by thapsigargin or bradykinin is functionally linked to a phosphoinositide-specific phospholipase D (PLD) activity in cultured vascular smooth muscle cells (VSMC), and that phosphatidic acid formed via PLD enhances sustained calcium entry in this cell type. These results suggest a regulatory role for PLD in store-operated Ca2+ entry in VSMC.     

c-Jun is required for TGF-β-mediated cellular migration via nuclear Ca²⁺ signaling

Tumor progression involves the acquisition of invasiveness through a basement membrane. The c-jun proto-oncogene is overexpressed in human tumors and has been identified at the leading edge of human breast tumors. TGF-β plays a bifunctional role in tumorigenesis and cellular migration. Although c-Jun and the activator protein 1 (AP-1) complex have been implicated in human cancer, the molecular mechanisms governing cellular migration via c-Jun and the role of c-Jun in TGF-β signaling remains poorly understood. Here, we analyze TGF-β mediated cellular migration in mouse embryo fibroblasts using floxed c-jun transgenic mice. We compared the c-jun wild type with the c-jun knockout cells through the use of Cre recombinase. Herein, TGF-β stimulated cellular migration and intracellular calcium release requiring endogenous c-Jun. TGF-β mediated Ca(2+) release was independent of extracellular calcium and was suppressed by both U73122 and neomycin, pharmacological inhibitors of the breakdown of PIP(2) into IP(3). Unlike TGF-β-mediated Ca(2+) release, which was c-Jun dependent, ATP mediated Ca(2+) release was c-Jun independent. These studies identify a novel pathway by which TGF-β regulates cellular migration and Ca(2+) release via endogenous c-Jun.     

Bradykinin stimulates a rise in cytosolic calcium in renal glomerular mesangial cells via a pertussis toxin insensitive pathway

Bradykinin elicits a complex response in the renal glomerulus which includes a reduction in the glomerular capillary ultrafiltration coefficient. To elucidate the biochemical mechanism of this response, we investigated calcium signalling in rat renal glomerular mesangial cells in culture using the calcium-sensitive fluorescent dye, Indo-1. Bradykinin was found to cause a concentration-dependent transient rise in cytosolic free calcium followed by a sustained slower secondary rise. The bradykinin response persisted with acute removal of extracellular calcium using EGTA, indicating that calcium entry from outside the cell did not mediate this primary response. Prolonged exposure to EGTA, which reduced intracellular stores, eliminated the calcium response to bradykinin but not to vasopressin, indicating differential sensitivity to intracellular calcium stores of these two hormonal responses. In agreement, prior stimulation with vasopressin significantly attenuated the response to bradykinin, but the converse did not occur. Aluminum fluoride and pertussis toxin were used to investigate the possible involvement of a guanyl nucleotide regulatory protein in signal transduction. Aluminum fluoride induced a transient rise in cytosolic calcium that was abrogated by prior exposure of the cells to pertussis toxin. This demonstrates the effectiveness of pertussis toxin and the presence of a calcium-signalling pathway susceptible to pertussis toxin in these cells. In contrast, the responses to bradykinin and vasopressin were unaffected by pertussis toxin. We conclude that bradykinin stimulates release of calcium from intracellular stores in glomerular mesangial cells via a pertussis toxin insensitive pathway. This mesangial response provides a direct biochemical basis for the bradykinin-induced fall in glomerular capillary ultrafiltration coefficient which has been observed in vivo.