Cloning and functional expression of a liver isoform of the small conductance Ca2+-activated K+ channel SK3

Small conductance Ca2+-activated K+ (SK) channels have been cloned from mammalian brain, but little is known about the molecular characteristics of SK channels in nonexcitable tissues. Here, we report the isolation from rat liver of an isoform of SK3. The sequence of the rat liver isoform differs from rat brain SK3 in five amino acid residues in the NH3 terminus, where it more closely resembles human brain SK3. SK3 immunoreactivity was detectable in hepatocytes in rat liver and in HTC rat hepatoma cells. Human embryonic kidney (HEK-293) cells transfected with liver SK3 expressed 10 pS K+ channels that were Ca2+ dependent (EC(50) 630 nM) and were blocked by the SK channel inhibitor apamin (IC(50) 0.6 nM); whole cell SK3 currents inactivated at membrane potentials more positive than -40 mV. Notably, the Ca2+ dependence, apamin sensitivity, and voltage-dependent inactivation of SK3 are strikingly similar to the properties of hepatocellular and biliary epithelial SK channels evoked by metabolic stress. These observations raise the possibility that SK3 channels influence membrane K+ permeability in hepatobiliary cells during liver injury.     

Differentiation dependent expression of TRPA1 and TRPM8 channels in IMR‐32 human neuroblastoma cells

TRPA1 and TRPM8 are transient receptor potential (TRP) channels involved in sensory perception. TRPA1 is a non‐selective calcium permeable channel activated by irritants and proalgesic agents. TRPM8 reacts to chemical cooling agents such as menthol. The human neuroblastoma cell line IMR‐32 undergoes a remarkable differentiation in response to treatment with 5‐bromo‐2‐deoxyuridine. The cells acquire a neuronal morphology with increased expression of N‐type voltage gated calcium channels and neurotransmitters. Here we show using RT‐PCR, that mRNA for TRPA1 and TRPM8 are strongly upregulated in differentiating IMR‐32 cells. Using whole cell patch clamp recordings, we demonstrate that activators of these channels, wasabi, allyl‐isothiocyanate (AITC) and menthol activate membrane currents in differentiated cells. Calcium imaging experiments demonstrated that AITC mediated elevation of intracellular calcium levels were attenuated by ruthenium red, spermine, and HC‐030031 as well as by siRNA directed against the channel. This indicates that the detected mRNA level correlate with the presence of functional channels of both types in the membrane of differentiated cells. Although the differentiated IMR‐32 cells responded to cooling many of the cells showing this response did not respond to TRPA1/TRPM8 channel activators (60% and 90% for AITC and menthol respectively). Conversely many of the cells responding to these activators did not respond to cooling (30%). This suggests that these channels have also other functions than cold perception in these cells. Furthermore, our results suggest that IMR‐32 cells have sensory characteristics and can be used to study native TRPA1 and TRPM8 channel function as well as developmental expression. J. Cell. Physiol. 221: 67–74, 2009. © 2009 Wiley‐Liss, Inc     

Identification and functional characterization of ion channels in CD34<Superscript>+</Superscript> hematopoietic stem cells from human peripheral blood

Hematopoietic stem cells (HSCs) are used therapeutically for hematological diseases and may also serve as a source for nonhematopoietic tissue engineering in the future. In other cell types, ion channels have been investigated as potential targets for the regulation of proliferation and differentiation. However, the ion channels of HSCs remain elusive. Here, we functionally characterized the ion channels of CD34⁺ cells from human peripheral blood. Using fluorescence-activated cell sorting, we confirmed that the CD34⁺ cells also express CD45 and CD133. In the CD34⁺/CD45⁺/CD133^high HSCs, RT-PCR of 58 ion channel mRNAs revealed the coexpression of Kv1.3, Kv7.1, Nav1.7, TASK2, TALK2, TWIK2, TRPC4, TRPC6, TRPM2, TRPM7, and TRPV2. Whole-cell patch clamp recordings identified voltage-gated K⁺ currents (putatively Kv1.3), pH-sensitive TASK2-like back-ground K⁺ currents, ADP-ribose-activated TRPM2 currents, temperature-sensitive TRPV2-like currents, and diacylglycerol-analogue-activated TRPC6-like currents. Our results lend new insight into the physiological role of ion channels in HSCs, the specific implications of which require further investigation.     

Induction of p18INK4c and its predominant association with CDK4 and CDK6 during myogenic differentiation.

Terminal cell differentiation involves permanent withdrawal from the cell division cycle. The inhibitors of cyclin-dependent kinases (CDKs) are potential molecules functioning to couple cell cycle arrest and cell differentiation. In murine C2C12 myoblast cells, G1 CDK enzymes (CDK2, CDK4, and CDK6) associate with four CDK inhibitors: p18INK4c, p19INK4d, p21, and p27Kip1. During induced myogenesis, p21 and its associated CDK proteins underwent an initial increase followed by a decrease as cells became terminally differentiated. The level of p27 protein gradually increased, but the amount of total associated CDK proteins remained unchanged. p19 protein decreased gradually during differentiation, as did its associated CDK4 protein. In contrast, p18 protein increased 50-fold, from negligible levels in proliferating myoblasts to clearly detectable levels within 8-12 h of myogenic induction. This initial rise was followed by a precipitous increase between 12 and 24 h postinduction, with p18 protein finally accumulating to its highest level in terminally differentiated cells. Induction of p18 correlated with increased and sequential complex formation--first increasing association with CDK6 and then with CDK4 over the course of myogenic differentiation. All of the CDK6 and half of the CDK4 were complexed with p18 in terminally differentiated C2C12 cells as well as in adult mouse muscle tissue. Finally, kinase activity of CDK2 and CDK4 decreases as C2C12 cells differentiate, whereas the CDK6 kinase activity is low in both proliferating myoblasts and differentiated myotubes. Our results indicate that p18 may play a critical role in causing and/or maintaining permanent cell cycle arrest associated with mature muscle formation.     

Contribution of a time-dependent and hyperpolarization-activated chloride conductance to currents of resting and hypotonically shocked rat hepatocytes

Hepatocellular Cl- flux is integral to maintaining cell volume and electroneutrality in the face of the many transport and metabolic activities that describe the multifaceted functions of these cells. Although a significant volume-regulated Cl- current (VRAC) has been well described in hepatocytes, the Cl- channels underlying the large resting anion conductance have not been identified. We used a combination of electrophysiological and molecular approaches to describe potential candidates for this conductance. Anion currents in rat hepatocytes and WIF-B and HEK293T cells were measured under patch electrode-voltage clamp. With K+-free salts of Cl- comprising the major ions externally and internally, hyperpolarizing steps between -40 and -140 mV activated a time-dependent inward current in hepatocytes. Steady-state activation was half-maximal at -63 mV and 28-38% of maximum at -30 to -45 mV, previously reported hepatocellular resting potentials. Gating was dependent on cytosolic Cl-, shifting close to 58 mV/10-fold change in Cl- concentration. Time-dependent inward Cl- currents and a ClC-2-specific RT-PCR product were also observed in WIF-B cells but not HEK293T cells. All cell types exhibited typical VRAC in response to dialysis with hypertonic solutions. DIDS (0.1 mM) inhibited the hepatocellular VRAC but not the inward time-dependent current. Antibodies against the COOH terminus of ClC-2 reacted with a protein between 90 and 100 kDa in liver plasma membranes. The results demonstrate that rat hepatocytes express a time-dependent inward Cl- channel that could provide a significant depolarizing influence in the hepatocyte.     

Growth potential of adult hepatocytes in mammals: highly replicative small hepatocytes with liver progenitor-like traits

The liver is one of the few organs that is capable of completely regenerating itself without using a stem cell population. When damaged, growth factors and cytokines are released, stimulating terminally differentiated adult hepatocytes and making them re-enter the cell cycle. We have been developing a series of studies on the growth potential of rat and human hepatocytes to identify a population of hepatocytes that is responsible for the regeneration of the injured liver. For this purpose, we established an appropriate culture method for hepatocytes by which growth and differentiation capacities are practically examined under various experimental conditions. This in vitro assay system allows us to identify small hepatocytes that are prominently replicative compared to large hepatocytes. Non-parenchymal cells play critical roles in the proliferation of small hepatocytes. These hepatocytes are present in both rat and human liver and are located in portal regions there. Phenotypic features were examined at morphological and gene/protein levels in detail, which showed the phenotypic plasticity in vitro. Mammalian liver includes a population of small hepatocytes in normal adults with a minute occupancy rate. We speculate that small hepatocytes play a role in regenerating the injured liver and in compensating for apoptotic hepatocytes in the physiological turnover of hepatocytes.     

Evidence for a hepatocellular lineage in a combined hepatocellular-cholangiocarcinoma of transitional type

A combined hepatocellular-cholangiocarcinoma (CHC) of transitional subtype and the surrounding cirrhotic liver tissue were investigated immunocytochemically by monoclonal antibodies specific for each of the keratin polypeptides 7, 8, 18 and 19. Different keratin subsets were found in different parts of the tumour. The hepatocellular component reveals keratins 8 and 18, with the bordering cells of trabecular formations additionally expressing keratins 7 and 19. The same keratins i.e. 7, 8, 18, 19 were found in normal bile duct epithelium as well as in cholangiocarcinomatous and transitional areas of hepatocellular and cholangiocellular differentiation. Normal hepatocytes express only keratin 8 and 18. In cirrhotic liver some modified hepatocytes additionally express keratin 7. When ductal transformation is observed in the marginal parts of portal tracts and fibrous septa the keratin polypeptide pattern mimics that of bile duct epithelium. The cholangiocellular metaplasia of hepatocytes observed here correlates well with findings in hepato-organogenesis and hepatocarcinogenesis and suggests that the transitional subtype of combined hepatocellular-cholangiocarcinoma is a variant of hepatocellular carcinoma.     

Evidence for a hepatocellular lineage in a combined hepatocellular-cholangiocarcinoma of transitional type

A combined hepatocellular-cholangiocarcinoma (CHC) of transitional subtype and the surrounding cirrhotic liver tissue were investigated immunocytochemically by monoclonal antibodies specific for each of the keratin polypeptides 7, 8, 18 and 19. Different keratin subsets were found in different parts of the tumour. The hepatocellular component reveals keratins 8 and 18, with the bordering cells of trabecular formations additionally expressing keratins 7 and 19. The same keratins i.e. 7, 8, 18, 19 were found in normal bile duct epithelium as well as in cholangiocarcinomatous and transitional areas of hepatocellular and cholangiocellular differentiation. Normal hepatocytes express only keratin 8 and 18. In cirrhotic liver some modified hepatocytes additionally express keratin 7. When ductal transformation is observed in the marginal parts of portal tracts and fibrous septa the keratin polypeptide pattern mimics that of bile duct epithelium. The cholangiocellular metaplasia of hepatocytes observed here correlates well with findings in hepato-organogenesis and hepatocarcinogenesis and suggests that the transitional subtype of combined hepatocellular-cholangiocarcinoma is a variant of hepatocellular carcinoma.     

Hepatogenic differentiation of mesenchymal stem cells in a rat model of thioacetamide-induced liver cirrhosis

Implantation of bone-marrow-derived MSCs (mesenchymal stem cells) has emerged as a potential treatment modality for liver failure, but in vivo differentiation of MSCs into functioning hepatocytes and its therapeutic effects have not yet been determined. We investigated MSC differentiation process in a rat model of TAA (thioacetamide)-induced liver cirrhosis. Male Sprague-Dawley rats were administered 0.04% TAA-containing water for 8 weeks, MSCs were injected into the spleen for transsplenic migration into the liver, and liver tissues were examined over 3 weeks. Ingestion of TAA for 8 weeks induced micronodular liver cirrhosis in 93% of rats. Injected MSCs were diffusely engrafted in the liver parenchyma, differentiated into CK19 (cytokeratin 19)- and thy1-positive oval cells and later into albumin-producing hepatocyte-like cells. MSC engraftment rate per slice was measured as 1.0-1.6%. MSC injection resulted in apoptosis of hepatic stellate cells and resultant resolution of fibrosis, but did not cause apoptosis of hepatocytes. Injection of MSCs treated with HGF (hepatocyte growth factor) in vitro for 2 weeks, which became CD90-negative and CK18-positive, resulted in chronological advancement of hepatogenic cellular differentiation by 2 weeks and decrease in anti-fibrotic activity. Early differentiation of MSCs to progenitor oval cells and hepatocytes results in various therapeutic effects, including repair of damaged hepatocytes, intracellular glycogen restoration and resolution of fibrosis. Thus, these results support that the in vivo hepatogenic differentiation of MSCs is related to the beneficial effects of MSCs rather than the differentiated hepatocytes themselves.     

EGF enhances the migration of cancer cells by up-regulation of TRPM7

Ion channels involved in the migration of tumor cells that is required for their invasion and metastasis. In this paper, we describe the interaction of TRPM7 channel and epidermal growth factor (EGF), an important player in cancer development in the migration of lung cancer cells. The TRPM7 currents in A549 cells were first characterized by means of electrophysiology, pharmacology and RNA interference. Removing Ca²⁺ from the extracellular solution not only potentiated a large inward current, but also abolished the outward rectification. 200 μM 2-APB inhibited the outward and the inward TRPM7 currents and at the same time restored the property of outward rectification. EGF greatly enhanced the migration of A549 cells, and also markedly up-regulated the membrane protein expression of TRPM7 and the amplitude of TRPM7 currents. Depressing the function of TRPM7 with RNA interference or pharmacological agents not only reversed the EGF-enhanced migration of A549 cells but also inhibited the basal migration of A549 cells in the absence of EGF. Thus it seems that TRPM7 plays a pivotal role in the migration of A549 cells induced by EGF and thus could be a potential therapeutic target in lung cancers.     

Dynamic expression of the cell adhesion molecule cell-CAM 105 in fetal and regenerating rat liver

Cell-CAM 105 is an integral cell surface glycoprotein that is involved in cell-cell adhesion of adult rat hepatocytes in vitro. In the present report we used a radio-immunoassay, a quantitative immunoblotting technique and immunofluorescence microscopy to investigate the expression of cell-CAM 105 in fetal and regenerating rat liver. In the fetal liver cell-CAM 105 did not appear until day 16 of the gestation, when it increased rapidly to reach the level found in adult liver, 3 weeks after birth. In liver regenerating after partial hepatectomy a transient decrease in the amount of cell-CAM 105 was observed in the plasma membranes of the hepatocytes. A significant decrease was observed as early as 12 h after partial hepatectomy, reaching a minimum by 3 days after the operation, corresponding to approx. 35% of the amount of cell-CAM 105 in normal liver. The amount then increased slowly and was back to the normal level by about 15 days after partial hepatectomy. The results indicate that cell-CAM 105 exerts its major function in terminally differentiated cells. An excellent correlation was seen between the kinetics of the expression of cell-CAM 105 and of reported changes of both enzymatic and organizational patterns of hepatocytes in regenerating and fetal liver. This suggests that cell-CAM 105 could be important for the development and maintenance of the cell-cell binding and organizational pattern characteristic of terminally differentiated hepatocytes.     

On potential interactions between non-selective cation channel TRPM4 and sulfonylurea receptor SUR1

The sulfonylurea receptor SUR1 associates with Kir6.2 or Kir6.1 to form K(ATP) channels, which link metabolism to excitability in multiple cell types. The strong physical coupling of SUR1 with Kir6 subunits appears exclusive, but recent studies argue that SUR1 also modulates TRPM4, a member of the transient receptor potential family of non-selective cation channels. It has been reported that, following stroke, brain, or spinal cord injury, SUR1 is increased in neurovascular cells at the site of injury. This is accompanied by up-regulation of a non-selective cation conductance with TRPM4-like properties and apparently sensitive to sulfonylureas, leading to the postulation that post-traumatic non-selective cation currents are determined by TRPM4/SUR1 channels. To investigate the mechanistic hypothesis for the coupling between TRPM4 and SUR1, we performed electrophysiological and FRET studies in COSm6 cells expressing TRPM4 channels with or without SUR1. TRPM4-mediated currents were Ca(2+)-activated, voltage-dependent, underwent desensitization, and were inhibited by ATP but were insensitive to glibenclamide and tolbutamide. These properties were not affected by cotransfection with SUR1. When the same SUR1 was cotransfected with Kir6.2, functional K(ATP) channels were formed. In cells cotransfected with Kir6.2, SUR1, and TRPM4, we measured K(ATP)-mediated K(+) currents and Ca(2+)-activated, sulfonylurea-insensitive Na(+) currents in the same patch, further showing that SUR1 controls K(ATP) channel activity but not TRPM4 channels. FRET signal between fluorophore-tagged TRPM4 subunits was similar to that between Kir6.2 and SUR1, whereas there was no detectable FRET efficiency between TRPM4 and SUR1. Our data suggest that functional or structural association of TRPM4 and SUR1 is unlikely.     

The role of transient receptor potential channel blockers in human gastric cancer cell viability

Transient receptor potential cation channel, subfamily M, receptor 7 (TRPM7) is a ubiquitous divalent-selective ion channel with its own kinase domain. Human gastric cancer cells express the TRPM7 channel, and the presence of this channel is essential for cell survival. Recent studies have suggested that 5-lipoxygenase (5-LOX) inhibitors are potent blockers of the TRPM7 channels. The aim of this study was to show the effects of 5-LOX inhibitors on the growth and survival of gastric cancer cells. Among 5-LOX inhibitors, nordihydroguaiaretic acid (NDGA), 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA861), and 3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol-2-yl]-2,2-dimethylpropanoic acid (MK886) were potent blockers of TRPM7-like currents in gastric cancer cells and also induced cell death. However, zileuton was ineffective in suppressing TRPM7-like current activity and inducing cell death. Moreover, a specific transient receptor potential cation channel, subfamily C, member 3 (TRPC3) inhibitor, a pyrazole compound (Pyr3), and a specific melastatin TRP (TRPM4) inhibitor, 9-phenanthrol, did not affect TRPM7-like currents or induce cell death. We conclude that TRPM7 has an important role in the growth and survival of gastric cancer cells and a likely potential target for the pharmacological treatment of gastric cancer.     

Transient receptor potential melastatin 1 (TRPM1) is an ion-conducting plasma membrane channel inhibited by zinc ions

TRPM1 is the founding member of the melastatin subgroup of transient receptor potential (TRP) proteins, but it has not yet been firmly established that TRPM1 proteins form ion channels. Consequently, the biophysical and pharmacological properties of these proteins are largely unknown. Here we show that heterologous expression of TRPM1 proteins induces ionic conductances that can be activated by extracellular steroid application. However the current amplitudes observed were too small to enable a reliable biophysical characterization. We overcame this limitation by modifying TRPM1 channels in several independent ways that increased the similarity to the closely related TRPM3 channels. The resulting constructs produced considerably larger currents after overexpression. We also demonstrate that unmodified TRPM1 and TRPM3 proteins form functional heteromultimeric channels. With these approaches, we measured the divalent permeability profile and found that channels containing the pore of TRPM1 are inhibited by extracellular zinc ions at physiological concentrations, in contrast to channels containing only the pore of TRPM3. Applying these findings to pancreatic β cells, we found that TRPM1 proteins do not play a major role in steroid-activated currents of these cells. The inhibition of TRPM1 by zinc ions is primarily due to a short stretch of seven amino acids present only in the pore region of TRPM1 but not of TRPM3. Combined, our data demonstrate that TRPM1 proteins are bona fide ion-conducting plasma membrane channels. Their distinct biophysical properties allow a reliable identification of endogenous TRPM1-mediated currents.     

The Poly(ADP-ribose) polymerase PARP-1 is required for oxidative stress-induced TRPM2 activation in lymphocytes

TRPM2 cation channels are widely expressed in the immune system and are thought to play a role in immune cell responses to oxidative stress. Patch clamp analyses suggest that TRPM2 channel activation can occur through a direct action of oxidants on TRPM2 channels or indirectly through the actions of a related group of adenine nucleotide 2nd messengers. However, the contribution of each gating mechanism to oxidative stress-induced TRPM2 activation in lymphocytes remains undefined. To better understand the molecular events leading to TRPM2 activation in lymphocytes, we analyzed oxidative stress-induced turnover of intracellular NAD, the metabolic precursor of adenine nucleotide 2nd messengers implicated in TRPM2 gating, and oxidative stress-induced TRPM2-mediated currents and Ca2+ transients in DT40 B cells. TRPM2-dependent Ca2+ entry did not influence the extent or time course of oxidative stress-induced turnover of NAD. Furthermore, expression of oxidative stress-activated poly(ADP-ribose) polymerases (PARPs) was required for oxidative stress-induced NAD turnover, TRPM2 currents, and TRPM2-dependent Ca2+ transients; no oxidant-induced activation of TRPM2 channels could be detected in PARP-deficient cells. Together, our results suggest that during conditions of oxidative stress in lymphocytes, TRPM2 acts as a downstream effector of the PARP/poly(ADP-ribose) glycohydrolase pathway through PARP-dependent formation of ADP-ribose.     

The helix-loop-helix inhibitor of differentiation (ID) proteins induce post-mitotic terminally differentiated Sertoli cells to re-enter the cell cycle and proliferate

Prior to puberty the Sertoli cells undergo active cell proliferation, and at the onset of puberty they become a terminally differentiated postmitotic cell population that support spermatogenesis. The molecular mechanisms involved in the postmitotic block of pubertal and adult Sertoli cells are unknown. The four known helix-loop-helix ID proteins (i.e., Id1, Id2, Id3, and Id4) are considered dominant negative regulators of cellular differentiation pathways and act as positive regulators of cellular proliferation. ID proteins are expressed at low levels by postpubertal Sertoli cells and are transiently induced by serum. The hypothesis tested was that ID proteins can induce a terminally differentiated postmitotic Sertoli cell to reenter the cell cycle if they are constitutively expressed. To test this hypothesis, ID1 and ID2 were stably integrated and individually overexpressed in postmitotic rat Sertoli cells. Overexpression of ID1 or ID2 allowed postmitotic Sertoli cells to reenter the cell cycle and undergo mitosis. The cells continued to proliferate even after 300 cell doublings. The functional markers of Sertoli cell differentiation such as transferrin, inhibin alpha, Sert1, and androgen binding protein (ABP) continued to be expressed by the proliferating Sertoli cells, but at lower levels. FSH receptor expression was lost in the proliferating Sertoli cell-Id lines. Some Sertoli cell genes, such as cyclic protein 2 (cathepsin L) and Sry-related HMG box protein-11 (Sox11) increase in expression. At no stage of proliferation did the cells exhibit senescence. The expression profile as determined with a microarray protocol of the Sertoli cell-Id lines suggested an overall increase in cell cycle genes and a decrease in growth inhibitory genes. These results demonstrate that overexpression of ID1 and ID2 genes in a postmitotic, terminally differentiated cell type have the capacity to induce reentry into the cell cycle. The observations are discussed in regards to potential future applications in model systems of terminally differentiated cell types such as neurons or myocytes.