Matrix stiffness-modulated proliferation and secretory function of the airway smooth muscle cells.

Shkumatov, Artem,Thompson, Michael,Choi, Kyoung M,Sicard, Delphine,Baek, Kwanghyun,Kim, Dong Hyun,Tschumperlin, Daniel J,Prakash, Y S,Kong, Hyunjoon American Physiological Society 2015 American journal of physiology. Lung cellular and Vol.308 No.11

<P>Multiple pulmonary conditions are characterized by an abnormal misbalance between various tissue components, for example, an increase in the fibrous connective tissue and loss/increase in extracellular matrix proteins (ECM). Such tissue remodeling may adversely impact physiological function of airway smooth muscle cells (ASMCs) responsible for contraction of airways and release of a variety of bioactive molecules. However, few efforts have been made to understand the potentially significant impact of tissue remodeling on ASMCs. Therefore, this study reports how ASMCs respond to a change in mechanical stiffness of a matrix, to which ASMCs adhere because mechanical stiffness of the remodeled airways is often different from the physiological stiffness. Accordingly, using atomic force microscopy (AFM) measurements, we found that the elastic modulus of the mouse bronchus has an arithmetic mean of 23.1 ± 14 kPa (SD) (median 18.6 kPa). By culturing ASMCs on collagen-conjugated polyacrylamide hydrogels with controlled elastic moduli, we found that gels designed to be softer than average airway tissue significantly increased cellular secretion of vascular endothelial growth factor (VEGF). Conversely, gels stiffer than average airways stimulated cell proliferation, while reducing VEGF secretion and agonist-induced calcium responses of ASMCs. These dependencies of cellular activities on elastic modulus of the gel were correlated with changes in the expression of integrin-관1 and integrin-linked kinase (ILK). Overall, the results of this study demonstrate that changes in matrix mechanics alter cell proliferation, calcium signaling, and proangiogenic functions in ASMCs.</P>

Ischemia induces regulator of G protein signaling 2 (RGS2) protein upregulation and enhances apoptosis in astrocytes

Endale, Mehari,Kim, Sung Dae,Lee, Whi Min,Kim, Sangseop,Suk, Kyoungho,Cho, Jae Youl,Park, Hwa Jin,Wagley, Yadav,Kim, Suk,Oh, Jae-Wook,Rhee, Man Hee American Physiological Society 2010 American journal of physiology. Cell physiology Vol.298 No.3

<P>Regulator of G protein signaling (RGS) family members, such as RGS2, interact with Gα subunits of heterotrimeric G proteins, accelerating the rate of GTP hydrolysis and attenuating the intracellular signaling triggered by the G protein-coupled receptor-ligand interaction. They are also reported to regulate G protein-effector interactions and form multiprotein signaling complexes. Ischemic stress-induced changes in RGS2 expression have been described in astrocytes, and these changes are associated with intracellular signaling cascades, suggesting that RGS2 upregulation may be an important mechanism by which astrocytes may regulate RGS2 function in response to physiological stress. However, information on the functional roles of stress-induced modulation of RGS2 protein expression in astrocyte function is limited. We report the role of ischemic stress in RGS2 protein expression in rat C6 astrocytoma cells and primary mouse astrocytes. A marked increase in RGS2 occurred after ischemic stress induced by chemicals (sodium azide and 2-deoxyglucose) or oxygen-glucose deprivation (OGD, real ischemia). RGS2 mRNA expression was markedly enhanced by 1 h of exposure to chemical ischemia or 6 h of OGD followed by 2 or 6 h of recovery, respectively. This enhanced expression in primary astrocytes and C6 cells was restored to baseline levels after 12 h of recovery from chemically induced ischemic stress or 4-6 h of recovery from OGD. RGS2 protein was also significantly expressed at 12-24 h of recovery from ischemic insult. Ischemia-induced RGS2 upregulation was associated with enhanced apoptosis. It significantly increased annexin V-positive cells, cleaved caspase-3, and enhanced DNA ladder formation and cell cycle arrest. However, a small interfering RNA (siRNA)-mediated RGS2 knockdown reversed the apoptotic cell death associated with ischemia-induced RGS2 upregulation. Upregulated RGS2 was significantly inhibited by SB-203580, a p38 MAPK inhibitor. Rottlerin, a potent inhibitor of PKCδ, completely abrogated the increased RGS2 expression. We also examine whether ischemia-induced RGS2-mediated apoptosis is affected by siRNA-targeted endogenous PKCδ downregulation or its phosphorylation. Although RGS2 upregulation was not affected, siRNA transfection significantly suppressed endogenous PKCδ mRNA and protein expressions. Ischemia-induced PKCδ phosphorylation and caspase-3 cleavage were dose dependently inhibited by PKCδ knockdown, and this endogenous PKCδ suppression reversed ischemia-induced annexin V-positive cells. This study suggests that ischemic stress increases RGS2 expression and that this condition contributes to enhanced apoptosis in C6 cells and primary astrocytes. The signaling it follows may involve PKCδ and p38 MAPK pathways.</P>

Orai1 and STIM1 in ER/PM junctions: roles in pancreatic cell function and dysfunction

Son, Aran,Park, Seonghee,Shin, Dong Min,Muallem, Shmuel American Physiological Society 2016 American journal of physiology. Cell physiology Vol.310 No.6

<P>Membrane contact sites (MCS) are critical junctions that form between the endoplasmic reticulum (ER) and membranes of various organelles, including the plasma membrane (PM). Signaling complexes, including mediators of Ca2+ signaling, are assembled within MCS, such as the ER/PM junction. This is most evident in polarized epithelial cells, such as pancreatic cells. Core Ca2+ signaling proteins cluster at the apical pole, the site of inositol 1,4,5-trisphosphate-mediated Ca2+ release and Orai1/transient receptor potential canonical-mediated store-dependent Ca2+ entry. Recent advances have characterized the proteins that tether the membranes at MCS and the role of these proteins in modulating physiological and pathological intracellular signaling. This review discusses recent advances in the characterization of Ca2+ signaling at ER/PM junctions and the relation of these junctions to physiological and pathological Ca2+ signaling in pancreatic acini.</P>

Silencing of MUC8 by siRNA increases P2Y₂-induced airway inflammation

Cha, Hee-Jae,Jung, Min-Su,Ahn, Do Whan,Choi, Jang-Kyu,Ock, Mee Sun,Kim, Kyung Soo,Yoon, Joo-Heon,Song, Eun Ju,Song, Kyoung Seob American Physiological Society 2015 American Journal of Physiology: Lung cellular and Vol.308 No.6

<P>Mucin hypersecretion and overproduction are frequent manifestations of respiratory disease. Determining the physiological function of airway mucin is presently considered more important than identifying the relevant signaling pathways. The lack of a full-length human mucin 8 (MUC8) cDNA sequence has hindered the generation of a Muc8 knockout mouse line. Thus, the precise physiological functions of MUC8 are unclear. Herein, we investigated the function of MUC8 using a small-interfering RNA (siRNA)-mediated genetic silencing approach in human airway epithelial cells. Herein, intracellular IL-1α production was stimulated by an ATP/P2Y<SUB>2</SUB> complex. While ATP/P2Y<SUB>2</SUB> increased IL-1α secretion in a time-dependent manner, treatment with P2Y<SUB>2</SUB>-specific siRNA significantly decreased IL-1α secretion. Moreover, ATP increased P2Y<SUB>2</SUB>-mediated upregulation of <I>MUC8</I> expression; however, IL-1α significantly decreased the extent to which ATP/P2Y<SUB>2</SUB> upregulated <I>MUC8</I> expression. Interestingly, treatment with MUC8-specific siRNA decreased the production of anti-inflammatory cytokines (TGF-β and IL-1 receptor antagonist) and increased the production of inflammatory cytokines (IL-1α and IL-6) in our system. In addition, siRNA-mediated knockdown of MUC8 expression dramatically increased the secretion of inflammatory chemokines and resulted in an approximately threefold decrease in cell chemotaxis. We propose that MUC8 may function as an anti-inflammatory mucin that participates in inflammatory response by attracting immune cells/cytokines to the site of inflammation. Our results provide new insight into the physiological function of MUC8 and enhance our understanding of mucin overproduction during airway inflammation.</P>

Linoleic acid stimulates gluconeogenesis via Ca2+/PLC, cPLA2, and PPAR pathways through GPR40 in primary cultured chicken hepatocytes.

Suh, Han Na,Huong, Huang Thi,Song, Chang Hun,Lee, Jang Hern,Han, Ho Jae American Physiological Society 2008 American journal of physiology. Cell physiology Vol.295 No.6

<P>Fatty acids serve vital functions as sources of energy, building materials for cellular structures, and modulators of physiological responses. Therefore, this study examined the effect of linoleic acid on glucose production and its related signal pathways in primary cultured chicken hepatocytes. Linoleic acid (double-unsaturated, long chain) increased glucose production in a dose (> or =10(-4) M)- and time (> or =8 h)-dependent manner. Both oleic acid (monounsaturated, long chain) and palmitic acid (saturated, long chain) also increased glucose production, whereas caproic acid (saturated, short chain) failed to increase glucose production. Linoleic acid increased G protein-coupled receptor 40 (GPR40; also known as free fatty acid receptor-1) protein expression and glucose production that was blocked by GPR40-specific small interfering RNA. Linoleic acid increased intracellular calcium concentration, which was blocked by EGTA (extracellular calcium chelator)/BAPTA-AM (intracellular calcium chelator), U-73122 (phospholipase C inhibitor), nifedipine, or methoxyverapamil (L-type calcium channel blockers). Linoleic acid increased cytosolic phospholipase A(2) (cPLA(2)) phosphorylation and the release of [(3)H]-labeled arachidonic acid. Moreover, linoleic acid increased the level of cyclooxygenase-2 (COX-2) protein expression, which stimulated the synthesis of prostaglandin E(2) (PGE(2)). The increase in PGE(2) production subsequently stimulated peroxisome proliferator-activated receptor (PPAR) expression, and MK-886 (PPAR-alpha antagonist) and GW-9662 (PPAR-delta antagonist) inhibited glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. In addition, linoleic acid-induced glucose production was blocked by inhibition of extracellular and intracellular calcium, cPLA(2), COX-2, or PPAR pathways. In conclusion, linoleic acid promoted glucose production via Ca(2+)/PLC, cPLA(2)/COX-2, and PPAR pathways through GPR40 in primary cultured chicken hepatocytes.</P>

Epigenetic regulation of integrin-linked kinase expression depending on adhesion of gastric carcinoma cells

Kim, Yong-Bae,Lee, Sung-Yul,Ye, Sang-Kyu,Lee, Jung Weon American Physiological Society 2007 American journal of physiology. Cell physiology Vol.292 No.2

<P>Cell adhesion to the extracellular matrix (ECM) regulates gene expressions in diverse dynamic environments. However, the manner in which gene expressions are regulated by extracellular cues is largely unknown. In this study, suspended gastric carcinoma cells showed higher basal and transforming growth factor-β1 (TGFβ1)-mediated acetylations of histone 3 (H3) and Lys<SUP>9</SUP>of H3 and levels of integrin-linked kinase (ILK) mRNA and protein than did fibronectin-adherent cells did. Moreover, the insignificant acetylation and ILK expression in adherent cells were recovered by alterations of integrin signaling and actin organization, indicating a connection between cytoplasmic and nuclear changes. Higher acetylations in suspended cells were correlated with associations between Smad4, p300/CBP, and Lys<SUP>9</SUP>-acetylated H3. Meanwhile, adherent cells showed more associations between HDAC3, Ski, and MeCP2. Chromatin immunoprecipitations with anti-acetylated H3, Lys<SUP>9</SUP>-acetylated H3, or p300/CBP antibody resulted in more coprecipitated ILK promoter, correlated with enhanced ILK mRNA and protein levels, in suspended cells. Moreover, ILK expression inversely regulated cell adhesion to ECM proteins, and its overexpression enhanced cell growth in soft agar. These observations indicate that cell adhesion and/or its related molecular basis regulate epigenetic mechanisms leading to a loss of ILK transcription, which in turn regulates cell adhesion property in a feedback linkage.</P>

TREK-2 (K2P10.1) and TRESK (K2P18.1) are major background K+ channels in dorsal root ganglion neurons.

Kang, Dawon,Kim, Donghee American Physiological Society 2006 American journal of physiology. Cell physiology Vol.291 No.1

<P>Dorsal root ganglion (DRG) neurons express mRNAs for many two-pore domain K(+) (K(2P)) channels that behave as background K(+) channels. To identify functional background K(+) channels in DRG neurons, we examined the properties of single-channel openings from cell-attached and inside-out patches from the cell bodies of DRG neurons. We found seven types of K(+) channels, with single-channel conductance ranging from 14 to 120 pS in 150 mM KCl bath solution. Four of these K(+) channels showed biophysical and pharmacological properties similar to TRESK (14 pS), TREK-1 (112 pS), TREK-2 (50 pS), and TRAAK (73 pS), which are members of the K(2P) channel family. The molecular identity of the three other K(+) channels could not be determined, as they showed low channel activity and were observed infrequently. Of the four K(2P) channels, the TRESK-like (14 pS) K(+) channel was most active at 24 degrees C. At 37 degrees C, the 50-pS (TREK-2 like) channel was the most active and contributed the most (69%) to the resting K(+) current, followed by the TRESK-like 14-pS (16%), TREK-1-like 112-pS (12%), and TRAAK-like 73-pS (3%) channels. In DRG neurons, mRNAs of all four K(2P) channels, as well as those of TASK-1 and TASK-3, were expressed, as judged by RT-PCR analysis. Our results show that TREKs and TRESK together contribute >95% of the background K(+) conductance of DRG neurons at 37 degrees C. As TREKs and TRESK are targets of modulation by receptor agonists, they are likely to play an active role in the regulation of excitability in DRG neurons.</P>

Bis deficiency results in early lethality with metabolic deterioration and involution of spleen and thymus.

Youn, Dong-Ye,Lee, Dong-Hyoung,Lim, Mi-Hyun,Yoon, Jung-Sook,Lim, Ji Hee,Jung, Seung Eun,Yeum, Chung Eun,Park, Cheol Whee,Youn, Ho-Joong,Lee, Jae-Seon,Lee, Seong-Beom,Ikawa, Masahito,Okabe, Masaru,Tsuj American Physiological Society 2008 AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND M Vol.295 No.6

<P>Bcl-2 interacting cell death suppressor (Bis), also known as Bag3 or CAIR-1, is involved in antistress and antiapoptotic pathways. In addition to Bcl-2, Bis binds to several proteins, suggesting it has diverse functions in normal and pathological conditions. To better define the physiological function of Bis in vivo, we developed bis-deficient mice with a cre-loxP system. Targeted disruption of exon 4 of the bis gene was demonstrated by Southern blotting and PCR, and Western blotting showed that no intact or truncated Bis protein was synthesized in bis(-/-) mice. While heterozygotes were fertile and appeared normal, Bis-deficient mice showed growth retardation and died by 3 wk after birth. The relative weight of the thymus and spleen was reduced and the total numbers of white blood cells, splenocytes, and thymocytes were significantly reduced compared with wild-type littermates. Serum profiles indicated significant hypoglycemia as well as decrease in triglyceride and cholesterol levels. Expression profiles of metabolic genes indicated that gluconeogenesis and beta-oxidation are activated in the liver of bis(-/-) mice. This activation, as well as a decrease in peripheral fat and an induction of fatty liver, appears to be an adaptive response to hypoglycemia. Our study reveals that the absence of Bis has considerable influences on postnatal growth and survival, possibly due to a nutritional impairment.</P>

Sodium arsenite induces orphan nuclear receptor SHP gene expression via AMP-activated protein kinase to inhibit gluconeogenic enzyme gene expression.

Chanda, Dipanjan,Kim, Sung-Jin,Lee, In-Kyu,Shong, Minho,Choi, Hueng-Sik American Physiological Society 2008 AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND M Vol.295 No.2

<P>Sodium arsenite has been demonstrated to alter the expression of genes associated with glucose homeostasis in tissues involved in the pathogenesis of type 2 diabetes; however, the underlying molecular mechanism has not been fully elucidated yet. In this study, we report that the sodium arsenite-induced gene expression of the small heterodimer partner (SHP; NR0B2), an atypical orphan nuclear receptor, regulates the expression of hepatic gluconeogenic genes. Sodium arsenite augments hepatic SHP mRNA levels in an AMP-activated protein kinase (AMPK)-dependent manner. Sodium arsenite activated AMPK and was shown to perturb cellular ATP levels. The arsenite-induced SHP mRNA level was blocked by adenoviral overexpression of dominant negative AMPK (Ad-dnAMPKalpha) or by the AMPK inhibitor compound C in hepatic cell lines. We demonstrated the dose-dependent induction of SHP mRNA levels by sodium arsenite and repressed the forskolin/dexamethasone-induced gene expression of the key hepatic gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). Ad-dnAMPKalpha blocked the repressive effects of arsenite-induced SHP on PEPCK and G6Pase. Sodium arsenite inhibited the promoter activity of PEPCK and G6Pase, and this repression was abolished by small interfering (si)RNA SHP treatments. The knockdown of SHP expression by oligonucleotide siRNA SHP or adenoviral siRNA SHP released the sodium arsenite-mediated repression of forskolin/dexamethasone-stimulated PEPCK and G6Pase gene expression in a variety of hepatic cell lines. Results from our study suggest that sodium arsenite induces SHP via AMPK to inhibit the expression of hepatic gluconeogenic genes and also provide us with a novel molecular mechanism of arsenite-mediated regulation of hepatic glucose homeostasis.</P>

Role of HIF-1α and VEGF in human mesenchymal stem cell proliferation by 17β-estradiol: involvement of PKC, PI3K/Akt, and MAPKs

Yun, Seung Pil,Lee, Min Yong,Ryu, Jung Min,Song, Chang Hun,Han, Ho Jae American Physiological Society 2009 American journal of physiology. Cell physiology Vol.296 No.2

<P>17β-Estradiol (E2) is a steroid hormone well known for its roles in the regulation of various cell functions. However, the precise role that E2plays in the proliferation of human mesenchymal stem cells (hMSCs) has not been completely elucidated. In the present study, we examined the effects of E2on cell proliferation and the related signaling pathways using hMSCs. We showed that E2, at ≥10<SUP>−9</SUP>M, significantly increased [<SUP>3</SUP>H]thymidine incorporation after 24 h of incubation, and E2also increased [<SUP>3</SUP>H]thymidine incorporation at >6 h. Also, E2significantly increased the percentage of the cell population in the S phase based on FACS analysis. Moreover, E2increased estrogen receptor (ER), PKC, phosphatidylinositol 3-kinase (PI3K)/Akt, and MAPK phosphorylation. Subsequently, these signaling molecules were involved in an E2-induced increase of [<SUP>3</SUP>H]thymidine incorporation. E2also increased hypoxia-inducible factor (HIF)-1α and VEGF protein levels. These levels of protein expression were inhibited by ICI-182,780 (10<SUP>−6</SUP>M, an ER antagonist), staurosporine and bisindolylmaleimide I (10<SUP>−6</SUP>M, a PKC inhibitor), LY-294002 (10<SUP>−6</SUP>M, a PI3K inhibitor), Akt inhibitor (10<SUP>−5</SUP>M), SP-600125 (10<SUP>−6</SUP>M, a SAPK/JNK inhibitor), and PD-98059 (10<SUP>−5</SUP>M, a p44/42 MAPKs inhibitor). In addition, HIF-1α small interfering (si)RNA and ICI-182,780 inhibited E2-induced VEGF expression and cell proliferation. VEGF siRNA also significantly inhibited E2-induced cell proliferation. In conclusion, E2partially stimulated hMSC proliferation via HIF-1α activation and VEGF expression through PKC, PI3K/Akt, and MAPK pathways.</P>