Calcium homeostatic pathways change with gestation in human myometrium

A rise in intracellular calcium is the primary trigger for contractile activity in pregnant human myometrium. It is hypothesized that key proteins involved in myometrial calcium homeostasis are gestationally regulated and play an important role in the preparation for labor. The aims of the study were to investigate the role of sarcoplasmic reticulum Ca ATPases (SERCAs) in regulating spontaneous contractile activity in myometrium, and to determine the expression of SERCA isoforms 2a and 2b, and the plasma membrane Ca ATPase (PMCA), at term and during labor. Western blot analysis demonstrated that the expression of SERCA 2a and 2b significantly increased in myometrium of women in labor compared with those not in labor. The augmentation of contractile activity in laboring myometrium in the presence of a SERCA 2 inhibitor, cyclopiazonic acid (CPA), demonstrated the functional significance of this observation. It is interesting that the application of CPA in the presence of a calcium-activated potassium channel inhibitor to term nonlabor myometrium mimicked the response of myometrium from women in active labor to CPA alone. We conclude that the activity of SERCA isoforms becomes increasingly important in the maintenance of regular contractile activity during labor and may compensate for the functional loss of other calcium control pathways at term.     

A spirochete surface protein uncouples store-operated calcium channels in fibroblasts: a novel cytotoxic mechanism

The cytotoxicity of infectious agents can be mediated by disruption of calcium signaling in target cells. Outer membrane proteins of the spirochete Treponema denticola, a periodontal pathogen, inhibit agonist-induced Ca(2+) release from internal stores in gingival fibroblasts, but the mechanism is not defined. We determined here that the major surface protein (Msp) of T. denticola perturbs calcium signaling in human fibroblasts by uncoupling store-operated channels. Msp localized in complexes on the cell surface. Ratio fluorimetry showed that in cells loaded with fura-2 or fura-C18, Msp induced cytoplasmic and near-plasma membrane Ca(2+) transients, respectively. Increased conductance was confirmed by fluorescence quenching of fura-2-loaded cells with Mn(2+) after Msp treatment. Calcium entry was blocked with anti-Msp antibodies and inhibited by chelating external Ca(2+) with EGTA. Msp pretreatment reduced the amplitude of [Ca(2+)](i) transients upon challenge with ATP or thapsigargin. In experiments using cells loaded with mag-fura-2 to report endoplasmic reticulum Ca(2+), Msp reduced Ca(2+) efflux from endoplasmic reticulum stores when ATP was used as an agonist. Msp alone did not induce Ca(2+) release from these stores. Msp inhibited store-operated influx of extracellular calcium following intracellular Ca(2+) depletion by thapsigargin and also promoted the assembly of subcortical actin filaments. This actin assembly was blocked by chelating intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester. The reduced amplitude of agonist-induced transients and inhibition of store-operated Ca(2+) entry due to Msp were reversed by latrunculin B, an inhibitor of actin filament assembly. Thus, Msp retards Ca(2+) release from endoplasmic reticulum stores, and it inhibits subsequent Ca(2+) influx by uncoupling store-operated channels. Actin filament rearrangement coincident with conformational uncoupling of store-operated calcium fluxes is a novel mechanism by which surface proteins and toxins of pathogenic microorganisms may damage host cells.     

Further insight into the roles of the glycans attached to human blood protein C inhibitor

Activation of T-cells triggers store-operated Ca²⁺ entry, which begins a signaling cascade leading to induction of appropriate gene expression and eventually lymphocyte proliferation and differentiation. The simultaneous enhancement of Fas ligand gene expression in activated cells allows the immune response to be limited by committing the activated cells to apoptosis. In apoptotic cells the store-operated calcium entry is significantly inhibited. It has been documented that moderate activation of Fas receptor may cause reversible inhibition of store-operated channels by ceramide released from hydrolyzed sphingomyelin. Here we show that activation of Fas receptor in T-cells results in caspase-dependent decrease of cellular STIM1 and Orai1 protein content. This effect may be responsible for the substantial inhibition of Ca²⁺ entry into Jurkat cells undergoing apoptosis. In turn, this inhibition might prevent overloading of cells with calcium and protect them against necrosis.     

Overexpression of transient receptor potential canonical type 1 (TRPC1) alters both store operated calcium entry and depolarization-evoked calcium signals in C2C12 cells

When the intracellular calcium stores are depleted, a Ca(2+) influx is activated to refill these stores. This store-operated Ca(2+) entry (SOCE) depends on the cooperation of several proteins as STIM1, Orai1, and, possibly, TRPC1. To elucidate this role of TRPC1 in skeletal muscle, TRPC1 was overexpressed in C2C12 cells and SOCE was studied by measuring the changes in intracellular Ca(2+) concentration ([Ca(2+)](i)). TRPC1 overexpression significantly increased both the amplitude and the maximal rate-of-rise of SOCE. When YM-58483, an inhibitor of TRPC1 was used, these differences were eliminated, moreover, SOCE was slightly suppressed. A decrease in the expression of STIM1 together with the downregulation of SERCA was confirmed by Western-blot. As a consequence, a reduction in maximal Ca(2+) uptake rate and a higher resting [Ca(2+)](i) following the Ca(2+) transients evoked by 120mM KCl were detected. Morphological changes also accompanied the overexpression of TRPC1. Differentiation of the myoblasts started later, and the myotubes were thinner in TRPC1-overexpressing cultures. For these changes the observed decrease in the nuclear expression of NFAT1 could be responsible. Our results suggest that enhanced expression of TRPC1 increases SOCE and has a negative effect on the STIM1-Orai1 system, indicating an interaction between these proteins.     

Expression of endomembrane calcium pumps in colon and gastric cancer cells. Induction of SERCA3 expression during differentiation

Calcium mobilization from the endoplasmic reticulum (ER) into the cytosol is a key component of several signaling networks controlling tumor cell growth, differentiation, or apoptosis. Sarco/endoplasmic reticulum calcium transport ATPases (SERCA-type calcium pumps), enzymes that accumulate calcium in the ER, play an important role in these phenomena. We report that SERCA3 expression is significantly reduced or lost in colon carcinomas when compared with normal colonic epithelial cells, which express this enzyme at a high level. To study the involvement of SERCA enzymes in differentiation, in this work differentiation of colon and gastric cancer cell lines was initiated, and the change in the expression of SERCA isoenzymes as well as intracellular calcium levels were investigated. Treatment of the tumor cells with butyrate or other established differentiation inducing agents resulted in a marked and specific induction of the expression of SERCA3, whereas the expression of the ubiquitous SERCA2 enzymes did not change significantly or was reduced. A similar marked increase in SERCA3 expression was found during spontaneous differentiation of post-confluent Caco-2 cells, and this closely correlated with the induction of other known markers of differentiation. Analysis of the expression of the SERCA3 alternative splice isoforms revealed induction of all three known iso-SERCA3 variants (3a, 3b, and 3c). Butyrate treatment of the KATO-III gastric cancer cells led to higher resting cytosolic calcium concentrations and, in accordance with the lower calcium affinity of SERCA3, to diminished ER calcium content. These data taken together indicate a defect in SERCA3 expression in colon cancers as compared with normal colonic epithelium, show that the calcium homeostasis of the endoplasmic reticulum may be remodeled during cellular differentiation, and indicate that SERCA3 constitutes an interesting new differentiation marker that may prove useful for the analysis of the phenotype of gastrointestinal adenocarcinomas.     

Capacitative calcium entry deficits and elevated luminal calcium content in mutant presenilin-1 knockin mice

Dysregulation of calcium signaling has been causally implicated in brain aging and Alzheimer's disease. Mutations in the presenilin genes (PS1, PS2), the leading cause of autosomal dominant familial Alzheimer's disease (FAD), cause highly specific alterations in intracellular calcium signaling pathways that may contribute to the neurodegenerative and pathological lesions of the disease. To elucidate the cellular mechanisms underlying these disturbances, we studied calcium signaling in fibroblasts isolated from mutant PS1 knockin mice. Mutant PS1 knockin cells exhibited a marked potentiation in the amplitude of calcium transients evoked by agonist stimulation. These cells also showed significant impairments in capacitative calcium entry (CCE, also known as store-operated calcium entry), an important cellular signaling pathway wherein depletion of intracellular calcium stores triggers influx of extracellular calcium into the cytosol. Notably, deficits in CCE were evident after agonist stimulation, but not if intracellular calcium stores were completely depleted with thapsigargin. Treatment with ionomycin and thapsigargin revealed that calcium levels within the ER were significantly increased in mutant PS1 knockin cells. Collectively, our findings suggest that the overfilling of calcium stores represents the fundamental cellular defect underlying the alterations in calcium signaling conferred by presenilin mutations.     

Conditional increase in SERCA2a protein is able to reverse contractile dysfunction and abnormal calcium flux in established diabetic cardiomyopathy

Diabetic cardiomyopathy is characterized by reduced cardiac contractility independent of vascular disease. A contributor to contractile dysfunction in the diabetic heart is impaired sarcoplasmic reticulum function with reduced sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a) pump activity, leading to disturbed intracellular calcium handling. It is currently unclear whether increasing SERCA2a activity in hearts with existing diabetic cardiomyopathy could still improve calcium flux and contractile performance. To test this hypothesis, we generated a cardiac-specific tetracycline-inducible double transgenic mouse, which allows for doxycycline (DOX)-based inducible SERCA2a expression in which DOX exposure turns on SERCA2a expression. Isolated cardiomyocytes and Langendorff perfused hearts from streptozotocin-induced diabetic mice were studied. Our results show that total SERCA2a protein levels were decreased in the diabetic mice by 60% compared with control. SERCA2a increased above control values in the diabetic mice after DOX. Dysfunctional contractility in the diabetic cardiomyocyte was restored to normal by induction of SERCA2a expression. Calcium transients from diabetic cardiomyocytes showed a delayed rate of diastolic calcium decay of 66%, which was reverted toward normal after SERCA2a expression induced by DOX. Global cardiac function assessed in the diabetic perfused heart showed diminished left ventricular pressure, rate of contraction, and relaxation. These parameters were returned to control values by SERCA2a expression. In conclusion, we have used mice allowing for inducible expression of SERCA2a and could demonstrate that increased expression of SERCA2a leads to improved cardiac function in mice with an already established diabetic cardiomyopathy in absence of detrimental effects.     

Phosphatase inhibition reveals a calcium entry pathway dependent on protein kinase A in thyroid FRTL-5 cells: comparison with store-operated calcium entry

Calcium entry through store-operated calcium channels is an important entry mechanism. In the present report we have described a novel calcium entry pathway that is independent of depletion of intracellular calcium stores. Treatment of the cells with the phosphatase inhibitor calyculin A (caly A), which blocked thapsigargin-evoked store-operated calcium entry (SOCE), induced a potent concentration-dependent calcium entry. In a calcium-free buffer, acute addition of caly A evoked a very modest increase in cytosolic free calcium ([Ca(2+)](i)). This increase was not from the agonist-mobilizable calcium stores, as the thapsigargin-evoked increase in [Ca(2+)](i) was unaltered in caly A-treated cells. The caly A-evoked calcium entry was not blocked by Gd(3+) or 2-APB, whereas SOCE was. Caly A enhanced the entry of barium, indicating that the increase in intracellular calcium was not the result of a decreased extrusion of calcium from the cytosol. Jasplakinolide and cytochalasin D had only marginal effects on calcium entry. The protein kinase A (PKA) inhibitor H-89 and an inhibitory peptide for PKA abolished the caly A-evoked entry of both calcium and barium. The SOCE was, however, enhanced in cells treated with H-89. In cells grown in the absence of thyrotropin (TSH), the caly A-evoked entry of calcium was smaller compared with cells grown in TSH-containing buffer. Stimulation of cells grown without TSH with forskolin or TSH restored the calyculin A-evoked calcium entry to that seen in cells grown in TSH-containing buffer. SOCE was decreased in these cells. Our results thus suggest that TSH, through the production of cAMP and activation of PKA, regulates a calcium entry pathway in thyroid cells. The pathway is distinctly different from the SOCE. As TSH is the main regulator of thyroid cells, we suggest that the novel calcium entry pathway participates in the regulation of basal calcium levels in thyroid cells.     

Progesterone receptor plays a major antiinflammatory role in human myometrial cells by antagonism of nuclear factor-kappaB activation of cyclooxygenase 2 expression

Spontaneous labor in women and in other mammals is likely mediated by a concerted series of biochemical events that negatively impact the ability of the progesterone receptor (PR) to regulate target genes that maintain myometrial quiescence. In the present study, we tested the hypothesis that progesterone/PR inhibits uterine contractility by blocking nuclear factor kappaB (NF-kappaB) activation and induction of cyclooxygenase-2 (COX-2), a contractile gene that is up-regulated in labor. To uncover mechanisms for regulation of uterine COX-2, immortalized human fundal myometrial cells were treated with IL-1beta +/- progesterone. IL-1beta alone caused a marked up-regulation of COX-2 mRNA, whereas treatment with progesterone suppressed this induction. This was also observed in human breast cancer (T47D) cells. In both cell lines, this inhibitory effect of progesterone was blocked by RU486. Using chromatin immunoprecipitation, we observed that IL-1beta stimulated recruitment of NF-kappaB p65 to both proximal and distal NF-kappaB elements of the COX-2 promoter; these effects were diminished by coincubation with progesterone. The ability of progesterone to inhibit COX-2 expression in myometrial cells was associated with rapid induction of mRNA and protein levels of inhibitor of kappaBalpha, a protein that blocks NF-kappaB transactivation. Furthermore, small interfering RNA-mediated ablation of both PR-A and PR-B isoforms in T47D cells greatly enhanced NF-kappaB activation and COX-2 expression. These effects were observed in the absence of exogenous progesterone, suggesting a ligand-independent action of PR. Based on these findings, we propose that PR may inhibit NF-kappaB activation of COX-2 gene expression and uterine contractility via ligand-dependent and ligand-independent mechanisms.     

Cell proliferation, calcium influx and calcium channels

Both increases in the basal cytosolic calcium concentration ([Ca²⁺]_cyt) and [Ca²⁺]_cyt transients play major roles in cell cycle progression, cell proliferation and division. Calcium transients are observed at various stages of cell cycle and more specifically during late G₁ phase, before and during mitosis. These calcium transients are mainly due to calcium release and reuptake by the endoplasmic reticulum (ER) and are observed over periods of hours in oocytes and mammalian cells. Calcium entry sustains the ER Ca²⁺ load and thereby helps to maintain these calcium transients for such a long period. Calcium influx also controls cell growth and proliferation in several cell types. Various calcium channels are involved in this process and the tight relation between the expression and activity of cyclins and calcium channels also suggests that calcium entry may be needed only at particular stages of the cell cycle. Consistent with this idea, the expression of l-type and T-type calcium channels and SOCE amplitude fluctuate along the cell cycle. But, as calcium influx regulates several other transduction pathways, the presence of a specific connection to trigger activation of proliferation and cell division in mammalian cells will be discussed in this review.     

Prostaglandin E2 Represses Interleukin 1 Beta-Induced Inflammatory Mediator Output from Pregnant Human Myometrial Cells Through the EP2 and EP4 Receptors

Inflammatory mediators, including prostaglandins, cytokines, and chemokines, are strongly implicated in the mechanism of human labor, though their precise roles remain unknown. Here we demonstrate that interleukin 1 beta (IL-1beta) significantly increased the expression and release of interleukin-8 (CXCL8), monocyte chemotactic protein-1 (CCL2), and granulocyte macrophage colony-stimulating factor (CSF2) by primary human myometrial cells. However, this effect was repressed by prostaglandin E(2) (PGE(2)). As PGE(2) can activate four distinct PGE(2) receptors (EP(1), EP(2), EP(3), and EP(4)) to elicit various responses, we sought to define the EP receptor(s) responsible for this repression. Using selective EP receptor agonists and a selective EP(4) antagonist, we show that PGE(2) mediates the repression of IL-1beta-induced release of CXCL8, CCL2, and CSF2 via activation of the EP(2) and EP(4) receptors. The use of siRNA gene-specific knockdown further confirmed a role for both receptors. Real-time RT-PCR demonstrated that EP(2) was the most highly expressed of all four EP receptors at the mRNA level in human myometrial cells, and immunocytochemistry showed that EP(2) protein is abundantly present throughout the cells. Interestingly, PGE(2) does not appear to reduce mRNA expression of CXCL8, CCL2, and CSF2. Our results demonstrate that PGE(2) can elicit anti-inflammatory responses via activation of the EP(2) and EP(4) receptors in lower segment term pregnant human myometrial cells. Further elucidation of the EP receptor-mediated signaling pathways in the pregnant human uterus may be beneficial for optimizing the maintenance of pregnancy, induction of labor or indeed treatment of preterm labor.     

Growth-dependent modulation of capacitative calcium entry in normal rat kidney fibroblasts

Normal rat kidney (NRK) fibroblasts have electrophysiological properties and intracellular calcium dynamics that are dependent upon their growth stage. In the present study we show that this differential behavior coincides with a differential calcium entry that can be either capacitative or non-capacitative. Confluent cells made quiescent by serum deprivation, which have a stable membrane potential near ? 70 mV and do not show spontaneous intracellular calcium oscillations, primarily exhibit the capacitative mechanism for calcium entry, also called store-operated calcium entry (SOCE). When the quiescent cells are grown to density-arrest in the presence of EGF as the sole polypeptide growth factor, these cells characteristically fire spontaneously repetitive calcium action potentials, which propagate throughout the whole monolayer and are accompanied by intracellular calcium transients. These density-arrested cells appear to exhibit in addition to SOCE also receptor-operated calcium entry (ROCE) as a mechanism for calcium entry. Furthermore we show that, in contrast to earlier studies, the employed SOCs and ROCs are permeable for both calcium and strontium ions. We examined the expression of the canonical transient receptor potential channels (Trpcs) that may be involved in SOCE and ROCE. We show that NRK fibroblasts express the genes encoding Trpc1, Trpc5 and Trpc6, and that the levels of their expression are dependent upon the growth stage of the cells. In addition we examined the growth stage dependent expression of the genes encoding Orai1 and Stim1, two proteins that have recently been shown to be involved in SOCE. Our results suggest that the differential expression of Trpc5, Trpc6, Orai1 and Stim1 in quiescent and density-arrested NRK fibroblasts is responsible for the difference in regulation of calcium entry between these cells. Finally, we show that inhibition or potentiation of SOCE and ROCE by pharmacological agents has profound effects on calcium dynamics in NRK fibroblasts.     

Evidence for a vesicle-mediated maintenance of store-operated calcium channels in a human embryonic kidney cell line

Direct microinjection of the clostridial neurotoxins botulinum neurotoxin A light chain or tetanus neurotoxin into cells of a human embryonic kidney cell line significantly reduced calcium entry after depletion of internal calcium stores by cyclopiazonic acid, a reversible inhibitor of the sarcoplasmic-endoplasmic reticular calcium-ATPases. Botulinum neurotoxin A light chain specifically hydrolyzes a synaptosomal-associated protein of 25 kilodaltons (SNAP-25), and tetanus neurotoxin specifically hydrolyzes synaptobrevin-2 (vesicle-associated membrane protein 2, VAMP-2) and cellubrevin (vesicle-associated membrane protein 3, VAMP-3). Since these substrate proteins are required for vesicle docking and fusion, inhibition of store-operated calcium entry by botulinum neurotoxin A light chain and tetanus neurotoxin supports a model in which vesicle fusion is a prerequisite for activation of store-operated calcium entry. Brefeldin A, a fungal metabolite that interferes with vesicle traffic, partially reduced calcium entry following store depletion. The size of the reserve pool of vesicles or parallel vesicle recycling pathways employing brefeldin A-sensitive and brefeldin A-insensitive ADP-ribosylation factors may explain the failure of brefeldin A to completely inhibit store-operated calcium entry.     

Localization and regulation of IL-1alpha in rat myometrium during late pregnancy and the postpartum period

Interleukin-1 (IL-1) has been implicated as a participant in preterm labor that is induced by bacterial infection. Previously, we showed that serotonin-induced production of IL-1alpha by myometrial smooth muscle cells in vitro is also essential for the synthesis of interstitial collagenase. It is therefore likely that IL-1alpha production in uterine tissues has implications for both the normal physiology of involution and for the pathophysiological mechanisms of preterm labor. The objective of this study was to characterize the serotonin-induced production of IL-1alpha by myometrial cultures in vitro and to assess the production of IL-1alpha and its relationship to collagenase production in vivo during pregnancy and the postpartum period. Immunohistochemistry demonstrated IL-1alpha protein in the nuclei and cytoplasm of serotonin-treated myometrial cells. IL-1alpha levels were decreased by treatment with progesterone or IL-1-receptor antagonist but were unaffected by lipopolysaccharide. Western analysis of myometrium from pregnant rats showed low levels of IL-1alpha during midpregnancy with increased concentrations at days 21 and 22 and postpartum. IL-1alpha mRNA levels also increased from days 15 to 22. Levels of mRNA for IL-1beta also increased, although to a lesser degree than IL-1alpha. Both mRNAs decreased postpartum. Conversely, mRNA for interstitial collagenase was barely detectable at term but increased postpartum. Together, these data show that serotonin stimulates IL-1alpha production in vitro and indicate that normal myometrium from pregnant rats is an identifiable source of IL-1 during late pregnancy. The findings are consistent with the possibility that myometrial IL-1alpha participates in normal labor as well as the postpartum production of interstitial collagenase.     

Tunicamycin desensitizes store-operated Ca entry to ATP and mitochondrial potential

Tunicamycin effect on thapsigargin-induced store-operated calcium entry was investigated. Ca²⁺ influx was stimulated by 50% upon exposure of Jurkat cells to tunicamycin. Moreover, tunicamycin efficiently prevented the inhibition of store-operated calcium entry caused by dissipation of mitochondrial membrane potential. Protective action of tunicamycin on store-operated Ca²⁺ entry was also partially preserved in Jurkat cells depleted of ATP, while Ca²⁺ entry into ATP-deprived cells grown in tunicamycin-free medium was almost completely inhibited. Tunicamycin-evoked changes in cellular Ca²⁺ fluxes coincided with decreased glycosylation of STIM1 protein. Although the latter observation is correlative and needs additional confirmation it may suggest that deglycosylation of STIM1 protein deprives store-operated calcium entry system of an important regulatory mechanism. This study suggests a novel mechanism of modulation of the activity of store-operated calcium channels in lymphoidal cells.