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Calcium and TRP Channels in Pulmonary Vascular Smooth Muscle Cell Proliferation

Judd W. Landsberg and Jason X.-J. Yuan

Department of Medicine, School of Medicine, University of California, San Diego, California 92103

	Abstract

 
Ca²⁺ is a major trigger for pulmonary vasoconstriction and a stimulus for pulmonary vascular smooth muscle cell proliferation. The transient receptor potential cation channels participate in regulating intracellular Ca²⁺ and thus vascular contractility and cell proliferation. Upregulation of genes encoding these channels is involved in the development of pulmonary hypertension.

	Introduction

Top Introduction Role of Ca2+ in... Role of Ca2+ in... Regulation of [Ca2+]cyt in... CCE in PASMC CCE is enhanced in... The molecular identity of... Upregulated TRPC expression in... Upregulated TRPC expression and... Summary References

 
Pulmonary hypertension is mediated to a large extent by plexogenic pulmonary arteriopathy. The dominant features of this arteriopathy include 1) sustained pulmonary vasoconstriction, 2) pulmonary arterial medial hypertrophy and muscularization of distal vessels due to smooth muscle cell hyperplasia and hypertrophy, and 3) obliterative intimal lesions in which smooth muscle cells predominate. Therefore, identifying the factors involved in triggering pulmonary vasoconstriction and promoting pulmonary artery smooth muscle cell (PASMC) proliferation is an area of active investigation. Pulmonary vascular tone and PASMC proliferation are both regulated by intracellular Ca²⁺ (6, 12, 14, 17, 20). An increase in cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_cyt) in PASMC induces both contraction and proliferation, thereby uniting the two fundamental derangements (pulmonary vasoconstriction and vascular medial hypertrophy) underlying severe pulmonary hypertension.

Vasoactive and mitogenic agonists trigger Ca²⁺ mobilization by activating inositol 1,4,5-trisphosphate (IP₃) receptors in the sarcoplasmic reticulum (SR) membrane, leading to SR Ca²⁺ release and subsequent depletion. This internal Ca²⁺ store depletion triggers a sustained Ca²⁺ influx known as capacitative Ca²⁺ entry (CCE). The two major functions of CCE are to refill the SR Ca²⁺ stores and to maintain adequate Ca²⁺ levels in the cytosol and nucleus in the face of high levels of mitogenic stimulation. The Ca²⁺ elevation associated with CCE occurs via activity of a special family of Ca²⁺ channels known as the store-operated Ca²⁺ channels (SOCs). These channels consist of hetero- or homotetrameric subunits encoded by the transient receptor potential (TRP) genes (19). Expression of TRP genes, function of SOCs, and amplitude of CCE are all involved in the regulation of pulmonary vascular tone and PASMC growth under physiological conditions (6, 17, 20). Furthermore, in PASMC isolated from patients with primary pulmonary hypertension (PPH), CCE is enhanced and TRP genes are upregulated, suggesting that increased expression of the SOC responsible for CCE may play an important role in the pathophysiology of PPH (6). This review focuses on the role of Ca²⁺ in PASMC proliferation, highlighting the importance of CCE, mediated by SOC and TRP gene expression, in maintaining the adequate intracellular Ca²⁺ required to stimulate and maintain proliferation.

	Role of Ca2+ in cell proliferation

Top Introduction Role of Ca2+ in... Role of Ca2+ in... Regulation of [Ca2+]cyt in... CCE in PASMC CCE is enhanced in... The molecular identity of... Upregulated TRPC expression in... Upregulated TRPC expression and... Summary References

 
Intracellular Ca²⁺ is a critical second messenger responsible for linking external stimuli to contraction, proliferation, and gene expression. Mitogenic stimulation leads to a rise in [Ca²⁺]_cyt due to Ca²⁺ release from internal stores and Ca²⁺ influx through sarcolemmal Ca²⁺ channels. A rise in [Ca²⁺]_cyt is transmitted by passive diffusion through nuclear pores in the nuclear envelope, thus increasing nuclear Ca²⁺ concentration ([Ca²⁺]_n) (8). Both elevated [Ca²⁺]_cyt and [Ca²⁺]_n, via an active Ca²⁺-calmodulin (CaM) complex, promote cell proliferation by stimulating quiescent cells to enter the cell cycle, as well as by driving proliferating cells through the cell cycle and mitosis. The activated Ca²⁺-CaM complex activates specific Ca²⁺-CaM-dependent kinases (e.g., CaM kinase IV) that promote phosphorylation of proteins, such as Ca²⁺/cAMP response element (CRE) binding protein (CREB, a nuclear Ca²⁺-responsive transcription factor) and Ras (a cytosolic Ca²⁺-responsive transcription factor), that are required for initiating and maintaining the cell cycle. In the cell cycle, the transition from G₀ to G₁ phase (i.e., from the resting state to the initiation of DNA synthesis), transition from G₁ to S phase (i.e., to the replication of nuclear DNA), and transition from G₂ to M (mitosis) phase as well as the entire mitosis phase are dependent on the activated Ca²⁺-CaM complex (1).

Beyond signal transduction, an adequate level of Ca²⁺ in intracellular stores, such as the SR, is crucial for normal cellular functions, including posttranslational modifications (e.g., correct folding, assembly, and glycosylation) of recombinant proteins. Therefore, maintenance of a high Ca²⁺ concentration in the SR ([Ca²⁺]_SR) also plays a critical role in optimizing protein and lipid synthesis and sorting. When the SR Ca²⁺ is depleted, the resultant disturbance of posttranslational modification of proteins may result in problematic protein trafficking, SR stress (3), growth arrest (15), and apoptosis (9). Indeed, removal of external Ca²⁺ and/or depletion of stored Ca²⁺ have been shown to induce growth arrest in vascular smooth muscle cells (6, 15).

	Role of Ca2+ in gene expression and cell growth

Top Introduction Role of Ca2+ in... Role of Ca2+ in... Regulation of [Ca2+]cyt in... CCE in PASMC CCE is enhanced in... The molecular identity of... Upregulated TRPC expression in... Upregulated TRPC expression and... Summary References

 
Elevated intracellular Ca²⁺, including a rise in [Ca²⁺]_cyt and/or in [Ca²⁺]_n, is the second messenger responsible for linking external mitogenic stimulus to gene expression in vascular smooth muscle cells. Expression of the immediate early genes that encode transcription factors like c-Fos and c-Jun, and of cell cycle regulators like cyclin A and E, can be stimulated by intracellular Ca²⁺ because the promoters of these genes contain the CRE (7, 8, 18). There are many Ca²⁺-sensitive transcription factors (e.g., c-Fos/c-Jun, Ras, cyclin A/B, CREB, proline-rich tyrosine kinase 2, NF-B) and signal transduction proteins (e.g., CREB kinase, CaM kinase IV, MAPK-activated protein kinase 2) that are involved in cell growth and proliferation (7, 8).

The magnitude, duration, and frequency of agonist- or mitogen-mediated rises in [Ca²⁺]_cyt and [Ca²⁺]_n determine what kinds of Ca²⁺-regulated genes are activated. A well-characterized Ca²⁺-activated gene is the c-fos gene; its promoter includes two elements that mediate the effect of Ca²⁺: 1) the serum response element that binds serum response factor and ternary complex factor, and 2) the CRE that binds the CREB. Induction of c-fos is a critical early event in cell growth. Expression of c-fos and c-jun proto-oncogenes is regulated by a sustained increase in [Ca²⁺]_cyt due to Ca²⁺ influx. A rise in [Ca²⁺]_cyt activates c-fos transcription via serum response element, whereas a rise in [Ca²⁺]_n activates c-fos transcription via CRE (7, 8). The differential activation of the c-fos promoter by the spatially distinct pools of Ca²⁺ indicates that local fluctuations of Ca²⁺ concentration may result in different transcriptional responses. Further studies indicate that the amplitude, duration, frequency, and spatial properties of the Ca²⁺ signals carried via distinct sarcolemmal Ca²⁺ channels (e.g., voltage-gated, receptor-operated, or store-operated) differ significantly. For example, Ca²⁺ influx through L-type voltage-gated Ca²⁺ channels efficiently activates CRE-dependent gene expression, but Ca²⁺ influx through N-methyl-D-aspartate (NMDA) receptors does not. An intriguing possibility is that the different Ca²⁺ channels associate preferentially with their specific Ca²⁺-activated effector molecules (2, 4, 5, 7, 8, 11), allowing this ubiquitous second messenger to have characteristic downstream effects.

	Regulation of [Ca2+]cyt in PASMC

Top Introduction Role of Ca2+ in... Role of Ca2+ in... Regulation of [Ca2+]cyt in... CCE in PASMC CCE is enhanced in... The molecular identity of... Upregulated TRPC expression in... Upregulated TRPC expression and... Summary References

 
In vascular smooth muscle cells, including PASMC, [Ca²⁺]_cyt can be increased by Ca²⁺ influx through sarcolemmal Ca²⁺ channels and by Ca²⁺ release from intracellular stores (mainly the SR). Both extracellular Ca²⁺ and intracellularly stored Ca²⁺ in the SR are significantly higher (5,000–20,000 times) than [Ca²⁺]_cyt. This enormous concentration gradient allows Ca²⁺ influx and release to occur without additional energy expenditure. [Ca²⁺]_cyt can be decreased by Ca²⁺ extrusion via the sarcolemmal Ca²⁺-Mg²⁺-ATPase and Na⁺/Ca²⁺ exchanger and by Ca²⁺ sequestration into the SR via the Ca²⁺-Mg²⁺-ATPase on the SR membrane (SERCA). In other words, cytoplasmic Ca²⁺ homeostasis is achieved by balancing the mechanisms that increase [Ca²⁺]_cyt (e.g., Ca²⁺ influx and release) with the mechanisms that decrease [Ca²⁺]_cyt (e.g., Ca²⁺ extrusion and sequestration) (Fig. 1).

View larger version (34K): [in this window] [in a new window]   FIGURE 1. Proposed mechanisms involved in the regulation of Ca2+ homeostasis in pulmonary artery smooth muscle cells (PASMC). Mitogenic agonists or growth factors bind to receptors, triggering the formation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). a: DAG activates receptor-operated channel (ROC)-mediated Ca2+ influx. b: IP3 activates IP3 receptor (IP3R)-mediated Ca2+ release. c: Subsequent depletion of Ca2+ from the sarcoplasmic reticulum (SR) in turn activates store-operated channel (SOC)-mediated Ca2+ influx. d: Membrane depolarization (e.g., by inhibition of Kv channel activity) leads to voltage-dependent Ca2+ channel (VDCC)-mediated Ca2+ influx. e: A high level of local cytoplasmic Ca2+ concentration ([Ca2+]cyt) is capable of triggering ryanodine receptor (RyR)-mediated Ca2+ release, a positive feedback mechanism known as Ca2+-induced Ca2+ release (CICR). The low [Ca2+]cyt under resting conditions is achieved and maintained mainly by active Ca2+ sequestration into the sarcoplasmic reticulum (SR) by the Ca2+-Mg2+-ATPase in the SR (SERCA) and Ca2+ extrusion by the Ca2+-Mg2+-ATPase in the plasma membrane.

Regulation of [Ca²⁺]_n is a matter of some debate and likely varies widely depending on cell type (8). In vascular smooth muscle cells, elevated [Ca²⁺]_cyt leads rapidly to elevated [Ca²⁺]_n by passive diffusion through nuclear pores. The nuclear pore protein has been shown to contain Ca²⁺ binding sites allowing conformational and, presumably, permeability change to occur on the basis of Ca²⁺ concentration (7, 8).

	CCE in PASMC

Top Introduction Role of Ca2+ in... Role of Ca2+ in... Regulation of [Ca2+]cyt in... CCE in PASMC CCE is enhanced in... The molecular identity of... Upregulated TRPC expression in... Upregulated TRPC expression and... Summary References

 
Depletion of Ca²⁺ from the SR triggers CCE. The SR Ca²⁺ store depletion can be achieved actively (by activating IP₃ receptors) or passively (by inhibiting SERCA). Exposure of PASMC to a mitogenic agonist in the absence of external Ca²⁺ increases IP₃ production, activates the IP₃ receptor, and ultimately depletes SR Ca²⁺ by inducing Ca²⁺ release. Passive store depletion occurs when SERCA is inhibited [e.g., by cyclopiazonic acid (CPA) or thapsigargin] and Ca²⁺ leaks out of the SR down a steep chemical or concentration gradient.

Figure 2 illustrates an example of how CCE is induced in PASMC by passive and active depletion of Ca²⁺ from the SR. As shown in Fig. 2A, CCE in PASMC can be observed as a rise in [Ca²⁺]_cyt occurring after passive store depletion with CPA. In the absence of extracellular Ca²⁺ (Fig. 2Aa), CPA, by blocking Ca²⁺ sequestration into the SR, induces a transient rise in [Ca²⁺]_cyt due to leakage of Ca²⁺ from the SR to the cytoplasm (Fig. 2Ab). The CPA-induced [Ca²⁺]_cyt transient declines back to the original baseline level after 5–7 min as the SR Ca²⁺ is depleted (Fig. 2Ac). Under these conditions, restoration of extracellular Ca²⁺ induces a further rise in [Ca²⁺]_cyt due to CCE (Fig. 2Ad), which is inhibited reversibly by the SOC blockers Ni²⁺ (Fig. 2Ae) and SKF-96365 (Fig. 2Ag). Removal of CPA from extracellular solution promotes cytosolic Ca²⁺ into the SR and reduces [Ca²⁺]_cyt (Fig. 2Ah).

View larger version (45K): [in this window] [in a new window]   FIGURE 2. A: schematic diagram and a representative record (tracing, middle) of [Ca2+]cyt change in PASMC showing the cellular events involved in capacitative Ca2+ entry (CCE) triggered by passive store depletion. When the cell is superfused with Ca2+-free solution (0Ca), [Ca2+]cyt is maintained at a very low level and the SR Ca2+ concentration ([Ca2+]SR) is very high due to an active Ca2+ uptake into the SR by SERCA (a). Application of cyclopiazonic acid (CPA) blocks SERCA and induces a transient rise in [Ca2+]cyt due to passive Ca2+ leakage from the SR to the cytosol according to its concentration gradient (b). The passive leakage soon (5–10 min) leads to store depletion and opening of SOC (c). Restoration of extracellular Ca2+ then results in a large increase in [Ca2+]cyt due to CCE (d). Functional blockade of SOC with Ni2+ (e) and SKF-96365 (SK, g) reversibly inhibits the CCE-induced increase in [Ca2+]cyt (f). Washout of extracellular CPA restores the function of SERCA and restores the intracellular Ca2+ milieu of a low [Ca2+]cyt and high [Ca2+]SR (h). B: CCE induces a contraction in isolated rat pulmonary artery (PA) rings triggered by active store depletion. In the absence of external Ca2+, the -adrenergic receptor agonist phenylephrine (PE, 10 µM) causes a transient contraction via IP3-mediated Ca2+ release, which is spontaneously terminated when internal stores are depleted. Restoration of extracellular Ca2+, despite the presence of the -receptor blocker phentolamine (Phen, 1 µM) and the VDCC blocker verapamil (Vp, 0.5 µM), gives rise to a sustained contraction due to CCE triggered by PE-induced active store depletion. C: whole cell SOC currents (ISOC) in PASMC induced by passive and active store depletion in PASMC. Cells were superfused with solutions containing 120 mM Na+ and 20 mM Ca2+ as charge carrier and were dialyzed with solutions containing 138 mM Cs+ (to eliminate K+ currents) and ~100 nM free Ca2+. The holding potential was set at 0 mV to inactivate voltage-gated cation (e.g., Na+ and Ca2+) and anion (e.g., Cl-) channels. Whole cell currents were elicited by 300-ms voltage steps from -120 to 0 mV before and after 15-min application of CPA (a) or PE (b) in the absence (CPA or PE) or presence (CPA+Ni or PE+La) of SOC blockers Ni2+ or La3+.

"...increase in [Ca²⁺]_cyt due to CCE is a critical signal transduction element ...."

Activity of SOCs is the major determinant of the amplitude of CCE, which, in turn, governs the magnitude of CCE-mediated contraction. The cation currents through store depletion-activated SOCs can be recorded by using patch clamp techniques in single PASMC (Fig. 2C). To record SOC currents (I_SOC) elicited by passive SR Ca²⁺ depletion, the cells are first superfused with solutions containing 120 Na⁺ and 20 mM Ca²⁺ and dialyzed with solutions containing 138 mM Cs⁺ (to eliminate K⁺ currents) and ~100 nM free Ca²⁺. The holding potential is set at 0 mV to inactivate voltage-gated cation (Na⁺ and Ca²⁺) and anion (Cl^-) channels. Whole cell currents are elicited by 300-ms voltage steps from -120 to 0 mV before and after application of CPA (Fig. 2Ca) or PE (Fig. 2Cb), which either passively or actively depletes Ca²⁺ from the SR within 10 min (see Fig. 2, A and B). Subtraction of the current recorded under control conditions from the current recorded during application of CPA or PE reveals that I_SOC is activated by either active or passive depletion of Ca²⁺ (Fig. 2C). Blockade of SOC with Ni²⁺ or La³⁺ reversibly reduces the amplitudes of I_SOC (Fig. 2C) and CCE (Fig. 2A) (6) and significantly inhibits CCE-mediated pulmonary vasoconstriction (12) and PASMC proliferation (6, 17, 20).

Together, these observations indicate that CCE can be induced by both active and passive depletion of stored Ca²⁺ in the SR. The increase in [Ca²⁺]_cyt due to CCE is a critical signal transduction element involved in smooth muscle contraction and PASMC proliferation.

	CCE is enhanced in PASMC during proliferation

Top Introduction Role of Ca2+ in... Role of Ca2+ in... Regulation of [Ca2+]cyt in... CCE in PASMC CCE is enhanced in... The molecular identity of... Upregulated TRPC expression in... Upregulated TRPC expression and... Summary References

 
Removal or chelation of extracellular Ca²⁺ inhibits PASMC growth in the presence of serum and growth factors (6, 15), suggesting that a constant Ca²⁺ influx is required for PASMC proliferation. Passive depletion of SR Ca²⁺ stores with CPA or thapsigargin further inhibits PASMC growth (6, 12, 15, 17, 20). The synergistically inhibitory effects of chelation of extracellular Ca²⁺ and depletion of stored Ca²⁺ in the SR on PASMC growth highlight the importance of both Ca²⁺ influx and adequate internal Ca²⁺ stores in PASMC proliferation.

When PASMC are growth arrested by incubation in a serum-free and growth factor-free medium, both the basal level of resting [Ca²⁺]_cyt (Fig. 3, A and Ba) and CPA-mediated CCE amplitude (Fig. 3, A and Bb) are much lower than in proliferating cells cultured in a medium containing 5% fetal bovine serum and growth factors. The rise in [Ca²⁺]_cyt due to CPA-mediated CCE is approximately threefold greater in proliferating PASMC than in growth-arrested cells and is insensitive to VDCC blockers and sensitive to the SOC blockers Ni²⁺ and SKF-96365 (see Fig. 2A) (6). Furthermore, the CPA-mediated transient increase in [Ca²⁺]_cyt in the absence of extracellular Ca²⁺ (which is mainly due to Ca²⁺ leakage from the SR to the cytoplasm, and its amplitude partially reflects [Ca²⁺]_SR) is approximately twofold greater in proliferating cells than in growth-arrested (or quiescent) PASMC. This indicates that Ca²⁺ concentration in the SR is also increased in proliferating PASMC (Fig. 3C). In addition to the fluorescence microscopy results, electrophysiological data also show that I_SOC, induced by passive store depletion with CPA, is significantly increased in proliferating PASMC compared with growth-arrested cells (6).

View larger version (46K): [in this window] [in a new window]   FIGURE 3. Resting [Ca2+]cyt and CCE are enhanced in PASMC during proliferation. A: representative records showing the changes in [Ca2+]cyt before, during, and after extracellular application of CPA in growth-arrested (left) and proliferating (right) PASMC. B: pseudocolor images showing the changes of [Ca2+]cyt in growth-arrested (a–d) and proliferating (a’–d’) PASMC in response to CPA (5 µM) in the absence and presence of extracellular Ca2+. Fura-2 fluorescence (F360) images show PASMC from which [Ca2+]cyt were measured (left). C: summarized data (means ± SE) showing the resting [Ca2+]cyt (left), the amplitude of CPA-mediated Ca2+ mobilization (middle), and the amplitude of CCE (right) in growth-arrested (blue bars) and proliferating (red bars) PASMC. **P < 0.01, *P < 0.05 vs. blue bars. Figure modified from Ref. 6, with permission.

The mechanism behind the enhanced CCE seen in proliferating PASMC likely involves upregulation of the genes responsible for encoding SOC (e.g., TRP genes). Exposure of PASMC to growth factors or mitogenic agonists activates tyrosine kinase-coupled receptors (e.g., PDGF receptor). The subsequent phosphorylation and activation of signal transduction proteins (e.g., protein kinases) and transcription factors (e.g.,signal transducer and activator of transcription, c-Jun) lead to upregulated TRP gene expression, increased SOC number, and, ultimately, enhanced CCE (Fig. 4) (20).

View larger version (31K): [in this window] [in a new window]   FIGURE 4. Schematic diagram depicting the role of upregulated canonical transient receptor potential family (TRPC) transcription and enhanced CCE in promoting PASMC proliferation. Activation of receptors by mitogenic stimulation leads to IP3 formation and subsequent IP3-mediated Ca2+ release. Repeated or constant mitogenic stimulation and Ca2+ release lead to store depletion, which in turn activates SOCs and triggers CCE. Ligand-receptor binding also mediates opening of ROCs via DAG and increases Ca2+ influx via ROCs. Upregulation of TRPC channel expression increases the number of SOCs and/or ROCs in the plasma membrane and enhances agonist-mediated Ca2+ influx via CCE and/or receptor-mediated Ca2+ entry. The subsequent increases in [Ca2+]cyt and nuclear Ca2+ concentration ([Ca2+]n) may promote PASMC proliferation by activating cytoplasmic signal transduction proteins [e.g., Ca2+/cAMP response element (CRE) binding protein (CREB) and mitogen-activated protein kinase] and transcription factors (e.g., c-Fos and c-Jun) and by moving quiescent cells into the cell cycle and propelling the proliferating cell through mitosis. In addition, a rise in [Ca2+]cyt and activation of STAT3 (e.g., via Ca2+-independent mechanism) may further enhance TRPC expression by upregulating c-Jun.

	The molecular identity of SOC in PASMC

Top Introduction Role of Ca2+ in... Role of Ca2+ in... Regulation of [Ca2+]cyt in... CCE in PASMC CCE is enhanced in... The molecular identity of... Upregulated TRPC expression in... Upregulated TRPC expression and... Summary References

The mammalian TRP family can be divided into three subfamilies: 1) TRP-canonical (TRPC) family (or short TRP channels), which is comprised of seven members, TRPC1–7; 2) TRP-vanilloid (TRPV) family (or Osm-9-like TRP channels), which consists of six members, TRPV1–6; and 3) TRP-melastatin (TRPM) family (or long TRP chanels), which contains eight members, TRPM1–8. There are three or more ankyrin domains in the NH₂-terminal cytosolic region of the short (TRPC) and Osm-9-like (TRPV) TRP channels and a proline-rich motif in the cytosolic COOH-terminal region in the vicinity of S6 of the short and long (TRPM) TRP channels. It has been shown that native SOCs are mainly heterotetramers comprised of different TRP isoforms. Sensitivity to store depletion may be mediated by the cytosolic COOH-terminal tail connected to the S6 transmembrane domain, but the mechanism linking store depletion to SOC activation is still unclear (19). The greatest homology between the subfamilies is seen in the S6 transmembrane domain, and the greatest variance is found in the COOH terminus. The subfamily members differ in their distribution throughout the body as well as in their mode of regulation.

Several human TRP homologs have been identified, including TRPC1, TRPC3, TRPC4, and TRPC6 in human lung tissues and pulmonary vascular smooth muscle and endothelial cells. The human TRP proteins likely encode ROCs as well as SOCs. Data from different cell types as well as experiments involving overexpression of specific TRP proteins have yielded conflicting results in attempting to correlate specific TRP isoforms with SOC properties. This may be explained by the heterotetrameric structure of the TRP channels. For example, a channel made up of two TRPC1, a TRPC3, and a TRPC6 protein may form a functional SOC activated by store depletion, whereas a channel made of two TRPC3 and two TRPC6 proteins may form a functional ROC activated predominantly by receptor-activated G proteins or diacylglycerol. By examining the gating property of overexpressed mammalian TRPC channels, numerous reports demonstrate that TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7 are activated in response to store depletion with SERCA inhibitors (e.g., thapsigargin, CPA) or ionophores (e.g., ionomycin). However, equally numerous reports indicate that TRPC channels overexpressed in mammalian cells are activated by PLC-coupled receptors and are not activated by store depletion (19). Therefore, the functional link between specific TRP gene products and agonist-mediated Ca²⁺ entry (e.g., CCE or receptor-mediated Ca²⁺ entry) remains an area for further study.

	Upregulated TRPC expression in human PASMC during proliferation

Top Introduction Role of Ca2+ in... Role of Ca2+ in... Regulation of [Ca2+]cyt in... CCE in PASMC CCE is enhanced in... The molecular identity of... Upregulated TRPC expression in... Upregulated TRPC expression and... Summary References

 
Human TRPC1 is widely distributed throughout the body, whereas TRPC6 is highly expressed in lung tissues and pulmonary arteries. Overexpression of TRPC6 in mammalian cells has been shown to enhance agonist-induced Ca²⁺ entry (10). Inhibition of TRPC6 expression with antisense oligonucleotides has been shown to inhibit agonist-mediated vascular tone in some experimental models (16). In proliferating human PASMC, TRPC1 and TRPC6 mRNA and protein expression are upregulated and the amplitude of I_SOC and CCE (induced by passive store depletion) are both enhanced. The increased mRNA expression of TRPC1 precedes the increase in cell number. Inhibition of either TRPC1 or TRPC6 gene expression with antisense oligonucleotides decreases CCE and attenuates PASMC proliferation (6, 17, 20). These data suggest that TRPC1 and TRPC6 may be involved in forming native SOCs responsible for CCE in human PASMC and, furthermore, that enhanced TRPC expression and CCE are a prerequisite for human PASMC proliferation.

In attempts to understand the mechanisms involved in the enhanced TRPC gene expression, I_SOC, and CCE seen in proliferating PASMC, we examined the role of STAT3 and c-Jun in rat PASMC proliferation (20). The tyrosine-phosphorylated STAT3 is a downstream signal transduction protein that is activated, via phosphorylation, upon ligand binding to the PDGF receptor. c-Jun is a transcription factor that stimulates gene expression and promotes cell growth. Treatment of PASMC with PDGF induces a transient increase (within 30–60 min) in protein levels of phosphorylated STAT3 (20), which is followed by a sustained increase (up to 48 h) in protein expression of c-Jun. Inhibition of c-Jun expression with specific antisense oligonucleotides prevents PDGF-induced upregulation of TRPC6, I_SOC, CCE, and proliferation (as measured by [³H]thymidine uptake). Overexpression of c-jun enhances TRPC6 protein expression in PASMC. These data suggest that growth factor-mediated TRPC upregulation may involve Ca²⁺-CaM-dependent kinase-mediated phosphorylation of STAT3 and upregulation of c-Jun (Fig. 4) (20).

	Upregulated TRPC expression and enhanced CCE in PASMC contribute to the development of PPH

Top Introduction Role of Ca2+ in... Role of Ca2+ in... Regulation of [Ca2+]cyt in... CCE in PASMC CCE is enhanced in... The molecular identity of... Upregulated TRPC expression in... Upregulated TRPC expression and... Summary References

 
As well as its essential role in normal PASMC proliferation, increased CCE, secondary to increased formation of SOCs by upregulated TRP genes, may be important in the development of pulmonary vascular remodeling in patients with PPH. Using PASMC isolated from patients undergoing lung transplant for PPH and secondary pulmonary hypertension (SPH), our laboratory has been able to compare the magnitude of CCE between PPH-PASMC and SPH-PASMC. When matched for pulmonary arterial pressure and pulmonary vascular resistance, growth-arrested PASMC from PPH patients demonstrate significantly higher resting [Ca²⁺]_cyt compared with cells from SPH patients. In addition, the magnitude of CCE, evoked by passive store depletion with CPA, is significantly greater in PASMC from PPH patients than in cells from SPH patients (6). At a molecular level, PPH is a heterogeneous disorder, with abnormal PASMC proliferation as its central theme. These data suggest that enhanced CCE, secondary to upregulated TRP genes, may be important in the pathogenesis of vascular remodeling in severe pulmonary hypertension and/or PPH. Accordingly, interruption of CCE at any point, from agents that downregulate TRPC gene expression to specific blockers for SOCs in PASMC, may prove beneficial in the development of therapeutic approaches for treatment of severe pulmonary hypertension.

	Summary

Top Introduction Role of Ca2+ in... Role of Ca2+ in... Regulation of [Ca2+]cyt in... CCE in PASMC CCE is enhanced in... The molecular identity of... Upregulated TRPC expression in... Upregulated TRPC expression and... Summary References

 
Elevated [Ca²⁺]_cyt is an important stimulus driving PASMC proliferation under both physiological and pathological conditions. By binding CaM, elevated cytoplasmic Ca²⁺ leads to the formation of the activated Ca²⁺-CaM complex. This complex is capable of activating several CaM kinases involved in the phosphorylation of signal transduction proteins and transcription factors responsible for promoting gene transcription and stimulating the PASMC to go through the cell cycle.

Mitogenic agonists stimulate PASMC growth by elevating [Ca²⁺]_cyt via Ca²⁺ influx and release. During PASMC proliferation, the essential resource of elevated [Ca²⁺]_cyt and [Ca²⁺]_n needs to be maintained by 1) increasing the number of functional Ca²⁺ channels in the plasma membrane, 2) enhancing the function of existing Ca²⁺ channels, and 3) maintaining the signal transduction between emptied SR and the plasmalemmal permeability to Ca²⁺. The TRPC family is a unique group of cation channels that are opened by both receptor activation and store depletion. Upregulation and functional enhancement of these channels have been implicated in both normal PASMC proliferation and in pulmonary vascular medial hypertrophy in patients with pulmonary arterial hypertension. Better defining the events involved in TRPC gene expression, SOC activation, and CCE regulation could lead to the development of new therapeutic approaches for abnormal PASMC proliferation in patients with severe pulmonary hypertension.

	Acknowledgments

 
This work was supported in part by grants from the National Heart, Lung, and Blood Institute (HL-64945, HL-54043, HL-66012, HL-69758, and HL-66941).

	References

Top Introduction Role of Ca2+ in... Role of Ca2+ in... Regulation of [Ca2+]cyt in... CCE in PASMC CCE is enhanced in... The molecular identity of... Upregulated TRPC expression in... Upregulated TRPC expression and... Summary References

 

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