Int J Clin Exp Pathol 2017;10(2):912-921 www.ijcep.com /ISSN:1936-2625/IJCEP0040599 Original Article Lipoteichoic acid of Streptococcus sanguinis induces the expression of cyclooxygenase-2 via MAPK signaling pathways in H9c2 cells Gloria Gutiérrez-Venegas, Israel Flores-Hernández Laboratorio de Bioquímica de la División de Estudios de Posgrado e Investigación de la Facultad de Odontología, Universidad Nacional Autónoma de México, México Received September 24, 2016; Accepted September 28, 2016; Epub February 1, 2017; Published February 15, 2017 Abstract: Bloodstream infections, including bacteremia and infective endocarditis, represent important medical conditions resulting in high mortality rates. Streptococcus sanguinis are dental plaque Gram-positive organisms as- sociated to infective endocarditis. Lipoteichoic acid, is a component of the outer membrane of Gram-positive bacte- ria and can cause septic shock. H9c2 cells were exposed to lipoteichoic acid in order to determine levels of nuclear factor (NF)-κB, mitogen activated protein kinase (MAPK) and cyclooxygenase-2. The results are presented as mean ± SE obtained from three independent experiments. A p value of < 0.05 was considered statistically significant. In this study, we characterized lipoteichoic acid signal-transduction mechanisms on H9c2 cardiomyoblasts. When H9c2 cells were exposed to lipoteichoic acid (15 μg/ml) they induced time-dependent augmented phoshorylation of MAPK; they also enhanced cytosolic and nuclear NF-κB levels in time-dependent manner. Furthermore, lipoteichoic acid significantly increased LTA-induced COX-2. LTA-mediated COX-2 expression was inhibited by PD98059, SP- 600125 and calphostin C. The present study showed that lipoteichoic acid obtained from Streptococcus sanguinis can increase cyclooxygenase-2 synthesis through protein kinase C, mitogen activated protein kinases and NF-κB activation in H9c2. Keywords: Lipoteichoic acid, cardiomyoblasts, mitogen activated protein kinases, nuclear factor-κB and cyclooxy- genase-2 Introduction The cell wall of Gram-positive bacteria encom- passes multiple functions during bacterial gr- Streptococcus group species are Gram-positive owth, such as maintaining bacterial cell integri- microorganisms present in dental plaque; they ty and shape as well as resisting internal turgor are associated to dental caries [1-3]. Bacteria pressure [12-14]. Moreover, the wall is com- may be seeded into the bloodstream through posed of peptidoglucan surrounding the cyto- brushing, chewing and oral surgery procedures. plasmic membrane and it includes glycopoly- In the blood bacteria may colonize endocardi- mers, such as teichoic acids, polysaccharides um or cardiac valves that have been damaged by congenital conditions or degenerative pro- and proteins. The most common types of tei- cesses resulting in infective endocarditis [4-6]. choic acids are comprised of either polyglycerol IE results fatal if not treated with antibiotics or phosphate or polyribitol phosphate chains of surgery. Streptococcus sanguinis is a member variable length that are substituted either with of the viridans group of streptococci and is a glycosylresidues or D-alanyl esters or both [15- primary colonizer of teeth. The viridans spe- 17]. Teichoic acids are covalently linked to pep- cies, particularly S. sanguinis are a leading tidoglycan or anchored in the cytoplasmic mem- cause of infective endocarditis [5-7]. Damage brane by their glycolipid moiety called lipoteic- is the result of the formation of sterile cardiac hoic acids, which are the main constituents of vegetations composed of platelets and fibrin Gram-positive bacteria surfaces, LTA have that can be colonized by certain bacteria during diverse biological functions such as autolysis, periods of bacteremia [8-11]. adhesion, biofilm formation and stimulation of Lipoteichoic acid promotes cyclooxygenase-2 expression in cardiomyocytes immune responses [15-20]. LTA binds to Toll- Cayman (Ann Arbor, MI). Antibodies were pur- like receptor 2 to transduce the LTA signal into chased from Santa Cruz Biotechnology (Santa the cytoplasm [21-23]. It also triggers MyD-88- Cruz, CA). dependent signaling pathway leading to NF-κB activation and can stimulate production of Cell culture cytokines, such as tumor necrosis factor-alpha H9c2 cardiomyoblasts, were cultured on 100 (TNF-α), COX-2, NOS-2 and interleukin-6 (IL-6), mm culture dishes in Dulbecco’s modified and excessive immune response to LTA results Eagle’s medium (DMEM) supplemented with in severe sepsis [24, 25]. Unlike Staphylococcus 100 μg/ml penicillin, 100 μg/ml, 2 mM gluta- aureus, which can cause severe sepsis [26, mine and 10% fetal bovine serum in humidified 27], some Gram-positive lactic acid bacteria do air (5% CO ) at 37°C. H9c2 cells were incubated not induce sepsis. However, the exact mecha- 2 in serum-free essential medium overnight nism of cytokine expression remains unclear. before LTA treatment. The intracellular signaling pathways through which LTA cause NOS expression have been Treatment of cells with LPS and inhibitors reported [28, 29], in macrophages through two separate pathways: the phosphatidylcholine- Calphostin C (1 μM) was used as specific inhibi- phospholipase/protein kinase C (PKC)/NF-κB tor of protein kinase C (PKC); PD98059 (10 μM) cascade and the tyrosine kinase/phosphati- was used as a specific inhibitor of MEK; dylinositol 3-kinase (PI3K)/AKT/p38 mitogen SB203580 (20 μM) was used as a specific activated protein kinase (MAPK)/NF-κB cas- inhibitor of p38 and SP600125 (10 μM) was cade [30]. Other studies have shown a positive used as a specific inhibitor of JNK. The inhibi- interaction between endogenous PGE2 synthe- tors were dissolved in DMSO at a stock concen- sis and NOS-2 expression both in vivo and in tration of 1 mM and stored at -20°C until use. vitro [31]. IL-1β plays a key role in host immune Cells were pretreated with inhibitors at the indi- regulation against different pathogens [32]. cated concentration or with DMSO vehicle for 1 The relevance of IL-1β has been reported in h before subjecting them to stimulation with septic arthritis, brain infections and periodon- LTA. tal disease [33, 34]. During the initial phases of infection IL-1β attracts phagocytic cells and Western blot assay increases lymphocyte [35]. Cells were plated onto 60 mm tissue-culture The results gathered in the present study dishes and stimulated with LTA or inhibitors for showed that LTA can cause activation of MAPK the indicated time period. The cells were then pathway by induction of COX-2 protein and for- washed with ice-cold PBS, lysed and then cen- mation of PGE2, resulting in the activation of trifuged at 13,000 g for 10 min. The whole cell MAPKs and NF-κB, which in turn induce IL-1β lysates were separated by 10% SDS-PAGE and expression. The signaling pathway explored in electro-transferred onto a PVDF membrane this study had a delayed onset, whereas two (Amersham Biosciences, Princeton, USA). After separate pathways which induce NF-κB activa- blocking with 5% skin milk in TBS containing tion in H9c2 cardiomyoblasts will be analyzed 0.1% Tween 20, the membrane was incubated in the present study. with antibodies against MAPKs, phosphorylat- ed MAPKs (1:1000), COX-2 (1:1000) followed Materials and methods by incubation with HRP-conjugated secondary antibiodies. The immune-reactive bands were Lipoteichoic acid (LTA obtained from Strepto- visualized with ECL reagents (Amersham coccus sanguinis), Trizma base, dithiothreitol Biosciences, Princeton, NJ, USA). (DTT), Dulbecco’s modified Eagle’s medium (DMEM), glycerol, phenylmethysulphonyl fluo- RT-PCR ride (PMSF), leupeptin and sodium dodecylsul- phate (SDS) were purchased from Sigma (St. H9c2 cells were plated onto six-well plates at Louis Mo, USA). Penicillin/streptomycin and 3×105 cells/ml and treated with LTA or inhibi- fetal bovine serum were purchase from Life tors at concentrations for the indicated time Technologies (Gaithersburg MD). A PGE2 en- period. Following treatment total RNA was iso- zyme immune-assay kit was obtained from lated using TRIzol reagent (Invitrogen, Carlsbad, 913 Int J Clin Exp Pathol 2017;10(2):912-921 Lipoteichoic acid promotes cyclooxygenase-2 expression in cardiomyocytes Figure 1. LTA-induced COX-2 expression in cardiomyoblasts. A: Cells were treated with vehicle or LTA at the indicated time periods. Cells were then harvested, COX-2 expression was determined by immunoblotting. Compiled results are shown at the bottom of the chart. Each column represents mean ± S.E.M. of at least three independent experi- ments. *P < 0.05 compared with control group. B: Cells were treated with vehicle or LTA at indicated concentrations for 6 h. After treatment, extent of COX-2 expression was determined by immunoblotting. Each column represented mean ± S.E.M. of at least three independent experiments. *P < 0.05 compared with control group. CA, USA), according to manufacturer’s instruc- way analysis of variance (ANOVA) followed by, tions. Total RNA was reverse-transcribed to when appropriate, the Newman-Keuls test was generate cDNA using OneStep kit (Invitrogen). used to determine the statistical significance of The sequence of each primer are as follows: the difference between means. A p value of < 5’-TTCAAATGAGATTGTGGGAAAATTGCT-3’ (cod- 0.05 was considered statistically significant. ing sense) and 5’-AGATCATCTCTGCCTGAGTAT- CTT-3’ (anticoding sense) derived from COX-2 Results gene; 5’-ACAGGGAAGTCTGAAGCACTAG-3’ (cod- ing sense) and 5’-CATGCAAGGAAGGGAACTCT- LTA induced