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The Low-Voltage-Activated Calcium Channel CAV3.1 Controls Proliferation of Human Pulmonary Artery Myocytes
Figure 1. Lung sections showing pulmonary arterioles in cross section (hematoxylin-eosin, magnification ). Scale bar ⫽ 50 m. Figure 3. HPV in Sprague-Dawley (SD) rats and spotted lethal (sl) rats following CBDL. The tracings of HPV in isolated lungs from Sham and CBDL rats. All data are from rats housed at the altitude of Denver.
The Low-Voltage-Activated Calcium Channel CAV3.1 Controls Proliferation of Human Pulmonary Artery Myocytes* D.M. Rodman, MD; J. Harral, MS; S. Wu, MD; J. West, PhD; M. Hoedt-Miller, MS; K.A. Reese, PhD; and K. Fagan, MD
(CHEST 2005; 128:581S–582S) ⫹⫹
hile Ca influx is essential for the activation of the W cell cycle machinery, the processes that allow Ca
⫹⫹
to enter the cell have not been clearly elucidated. Elec-
Figure 2. Top, A: pulmonary Western blots (upper panel) and corresponding densitometry (lower panel) for ETa receptor (white and gray columns) and ETb receptor (crosshatch and black columns). Bottom, B: pulmonary ET-1 levels measured by enzyme-linked immunosorbent assay (n ⫽ 3 to 4 for all groups). www.chestjournal.org
*From the Center for Genetic Lung Disease (Drs. Rodman, West, Reese, and Fagan, Mr. Harral, and Mr. Hoedt-Miller), Division of Pulmonary Sciences and Critical Care Medicine (Dr. Wu), University of South Alabama, Mobile, AL. This work was supported by grants HL48038, HL57282, and HL14985. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. org/misc/reprints.shtml). Correspondence to: K.A. Reese, PhD, 4200 E Ninth Ave, B133, Denver, CO 80262; e-mail: [email protected] CHEST / 128 / 6 / DECEMBER, 2005 SUPPLEMENT
581S
trophysiologic and molecular studies have identified multiple Ca⫹⫹ channel genes expressed in mammalian cells. CaV3.x gene family members, encoding low voltage-activated or T-type Ca⫹⫹ channels, were first identified in the CNS and subsequently in nonneuronal tissue. There are conflicting reports describing a potential role for T-type Ca⫹⫹ channels in controlling the cell cycle and proliferation. In the present study, using a whole-cell patch clamp, quantitative reverse transcriptase polymerase chain reaction, and immunocytochemistry we found that CaV3.1 was the predominant CaV3.x channel expressed in early passage human pulmonary artery cells in vitro and in the media of human pulmonary arteries in vivo. CaV3.x messenger RNA expression was highest on reentry to the cell cycle. Selective blockade of CaV3.1 expression with small-interfering RNA and pharmacologic blockade of T-type currents completely inhibited proliferation in response to a 5% serum and prevented cell-cycle entry.
Conclusion T-type voltage-dependent Ca⫹⫹ channels encoded by the CaV3.1 gene are required for cell-cycle progression and proliferation of human pulmonary arterial smooth muscle cells. This proliferation could be important for the development of pulmonary hypertension.
Hypoxia-Induced Alterations in Protein Kinase C Signaling Result in Augmented Fibroblast Proliferation*
activated protein kinase phosphatase 1 (MKP1), an important terminator of ERK activation, has been identified as a hypoxia-inducible gene. However, the role of MKP1 in the hypoxia-induced proliferation of fibroblasts is unknown. Since hypoxia activates the PKC isozyme in many cell types, we hypothesized that PKC will control the hypoxic proliferation of vascular fibroblasts through the regulation of MKP1, and thus ERK1/2, and that this PKC-mediated mechanism will be different in fibroblasts from control animals vs chronically hypoxic animals. Using fibroblasts isolated from the pulmonary artery adventitia of neonatal control and chronically hypoxic calves, we found that, hypoxia, in the absence of any comitogens, induced the activation of PKC. The treatment of control cells with either a myristoylated pseudosubstrate peptide inhibitor or antisense oligonucleotides of PKC induced a marked increase in hypoxia-induced proliferation, significant upregulation of ERK1/2 activation, and the blockade of hypoxia-stimulated increases in MKP1 levels. In contrast, in fibroblasts from chronically hypoxic calves, PKC inhibition blocked hypoxia-induced proliferation as well as ERK1/2 activation. MKP1 was not up-regulated in these cells under hypoxic conditions. These results demonstrate that chronic hypoxic exposure induces changes in the downstream targets of PKC in vascular adventitial fibroblasts. In normal fibroblasts, PKC-mediated MKP1 activation acts to limit cell proliferation. However, following chronic hypoxic exposure, PKC-regulated activation of MKP1 is lost, resulting in fibroblasts with heightened proliferative capabilities.
Abbreviations: ERK ⫽ extracellular signal-regulated kinase; MKP1 ⫽ mitogen-activated protein kinase phosphatase 1; PKC ⫽ protein kinase C
Role of Rho in the Increased Migration of Pulmonary Artery Smooth Muscle Cells Observed in the Neprilysin Knockout Mouse
hypoxic exposure induces marked remodeling C hronic in the adventitial compartment of the pulmonary
Vijaya Karoor, Ph.D; Sandra J. Walchak; York E. Miller, MD; and Edward C. Dempsey, MD, FCCP
Megan Short; Stephanie Fox; Kurt R. Stenmark; and Mita Das
(CHEST 2005; 128:582S)
artery, which is due, at least in part, to increased proliferation of the resident fibroblast populations. We have demonstrated that hypoxia can stimulate the proliferation of adventitial fibroblasts through the activation of protein kinase C (PKC) and extracellular signal-regulated kinases (ERKs) 1/2 pathways and that those proliferative responses are greater in fibroblasts derived from chronically hypoxic animals than those from the controls. Mitogen*From the Department of Pediatrics, University of Colorado Health Sciences Center, Denver, CO. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. org/misc/reprints.shtml). Correspondence to: Mita Das, the Department of Pediatrics, University of Colorado Health Sciences Center, Denver, CO 80262 582S
(CHEST 2005; 128:582S–583S) Abbreviations: KO ⫽ knockout; NEP ⫽ neprilysin; PASMC ⫽ pulmonary areterial smooth muscle cell; PKC ⫽ protein kinase C; PI3K ⫽ phosphatidyl inositol-3-kinase
*From the Cardiovascular Pulmonary Research Laboratory, University of Colorado Health sciences Center, Denver, CO. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. org/misc/reprints.shtml). Correspondence to: Edward C. Dempsey, MD, FCCP, UCHSC, Box B133, 4200 East 9th St, Denver, CO 80262; e-mail: [email protected]
47th Annual Thomas L. Petty Lung Conference: Cellular and Molecular Pathobiology of Pulmonary Hypertension
The Low-Voltage-Activated Calcium Channel CAV3.1 Controls Proliferation of Human Pulmonary Artery Myocytes* D.M. Rodman, MD; J. Harral, MS; S. Wu, MD; J. West, PhD; M. Hoedt-Miller, MS; K.A. Reese, PhD; and K. Fagan, MD
(CHEST 2005; 128:581S–582S) ⫹⫹
hile Ca influx is essential for the activation of the W cell cycle machinery, the processes that allow Ca
⫹⫹
to enter the cell have not been clearly elucidated. Elec-
Figure 2. Top, A: pulmonary Western blots (upper panel) and corresponding densitometry (lower panel) for ETa receptor (white and gray columns) and ETb receptor (crosshatch and black columns). Bottom, B: pulmonary ET-1 levels measured by enzyme-linked immunosorbent assay (n ⫽ 3 to 4 for all groups). www.chestjournal.org
*From the Center for Genetic Lung Disease (Drs. Rodman, West, Reese, and Fagan, Mr. Harral, and Mr. Hoedt-Miller), Division of Pulmonary Sciences and Critical Care Medicine (Dr. Wu), University of South Alabama, Mobile, AL. This work was supported by grants HL48038, HL57282, and HL14985. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. org/misc/reprints.shtml). Correspondence to: K.A. Reese, PhD, 4200 E Ninth Ave, B133, Denver, CO 80262; e-mail: [email protected] CHEST / 128 / 6 / DECEMBER, 2005 SUPPLEMENT
581S
trophysiologic and molecular studies have identified multiple Ca⫹⫹ channel genes expressed in mammalian cells. CaV3.x gene family members, encoding low voltage-activated or T-type Ca⫹⫹ channels, were first identified in the CNS and subsequently in nonneuronal tissue. There are conflicting reports describing a potential role for T-type Ca⫹⫹ channels in controlling the cell cycle and proliferation. In the present study, using a whole-cell patch clamp, quantitative reverse transcriptase polymerase chain reaction, and immunocytochemistry we found that CaV3.1 was the predominant CaV3.x channel expressed in early passage human pulmonary artery cells in vitro and in the media of human pulmonary arteries in vivo. CaV3.x messenger RNA expression was highest on reentry to the cell cycle. Selective blockade of CaV3.1 expression with small-interfering RNA and pharmacologic blockade of T-type currents completely inhibited proliferation in response to a 5% serum and prevented cell-cycle entry.
Conclusion T-type voltage-dependent Ca⫹⫹ channels encoded by the CaV3.1 gene are required for cell-cycle progression and proliferation of human pulmonary arterial smooth muscle cells. This proliferation could be important for the development of pulmonary hypertension.
Hypoxia-Induced Alterations in Protein Kinase C Signaling Result in Augmented Fibroblast Proliferation*
activated protein kinase phosphatase 1 (MKP1), an important terminator of ERK activation, has been identified as a hypoxia-inducible gene. However, the role of MKP1 in the hypoxia-induced proliferation of fibroblasts is unknown. Since hypoxia activates the PKC isozyme in many cell types, we hypothesized that PKC will control the hypoxic proliferation of vascular fibroblasts through the regulation of MKP1, and thus ERK1/2, and that this PKC-mediated mechanism will be different in fibroblasts from control animals vs chronically hypoxic animals. Using fibroblasts isolated from the pulmonary artery adventitia of neonatal control and chronically hypoxic calves, we found that, hypoxia, in the absence of any comitogens, induced the activation of PKC. The treatment of control cells with either a myristoylated pseudosubstrate peptide inhibitor or antisense oligonucleotides of PKC induced a marked increase in hypoxia-induced proliferation, significant upregulation of ERK1/2 activation, and the blockade of hypoxia-stimulated increases in MKP1 levels. In contrast, in fibroblasts from chronically hypoxic calves, PKC inhibition blocked hypoxia-induced proliferation as well as ERK1/2 activation. MKP1 was not up-regulated in these cells under hypoxic conditions. These results demonstrate that chronic hypoxic exposure induces changes in the downstream targets of PKC in vascular adventitial fibroblasts. In normal fibroblasts, PKC-mediated MKP1 activation acts to limit cell proliferation. However, following chronic hypoxic exposure, PKC-regulated activation of MKP1 is lost, resulting in fibroblasts with heightened proliferative capabilities.
Abbreviations: ERK ⫽ extracellular signal-regulated kinase; MKP1 ⫽ mitogen-activated protein kinase phosphatase 1; PKC ⫽ protein kinase C
Role of Rho in the Increased Migration of Pulmonary Artery Smooth Muscle Cells Observed in the Neprilysin Knockout Mouse
hypoxic exposure induces marked remodeling C hronic in the adventitial compartment of the pulmonary
Vijaya Karoor, Ph.D; Sandra J. Walchak; York E. Miller, MD; and Edward C. Dempsey, MD, FCCP
Megan Short; Stephanie Fox; Kurt R. Stenmark; and Mita Das
(CHEST 2005; 128:582S)
artery, which is due, at least in part, to increased proliferation of the resident fibroblast populations. We have demonstrated that hypoxia can stimulate the proliferation of adventitial fibroblasts through the activation of protein kinase C (PKC) and extracellular signal-regulated kinases (ERKs) 1/2 pathways and that those proliferative responses are greater in fibroblasts derived from chronically hypoxic animals than those from the controls. Mitogen*From the Department of Pediatrics, University of Colorado Health Sciences Center, Denver, CO. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. org/misc/reprints.shtml). Correspondence to: Mita Das, the Department of Pediatrics, University of Colorado Health Sciences Center, Denver, CO 80262 582S
(CHEST 2005; 128:582S–583S) Abbreviations: KO ⫽ knockout; NEP ⫽ neprilysin; PASMC ⫽ pulmonary areterial smooth muscle cell; PKC ⫽ protein kinase C; PI3K ⫽ phosphatidyl inositol-3-kinase
*From the Cardiovascular Pulmonary Research Laboratory, University of Colorado Health sciences Center, Denver, CO. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. org/misc/reprints.shtml). Correspondence to: Edward C. Dempsey, MD, FCCP, UCHSC, Box B133, 4200 East 9th St, Denver, CO 80262; e-mail: [email protected]
47th Annual Thomas L. Petty Lung Conference: Cellular and Molecular Pathobiology of Pulmonary Hypertension