_:b493961925 . _:b16174313 . _:b16174252 "The pathogenic progression involves glial activation and TLR2 signaling stimulated by LTA, which are linked to inflammatory neurodegeneration [>>9<<-11]. Additionally, LTA exhibits detrimental effects on brain cellular functions, including induction of apoptosis, production of oxidative stresses and disruption of BBB following group B Streptococcus or Staphylococcus aureus challenge" . _:b16174230 "NADPH oxidase (Nox) is considered to be a major source of ROS in several physiological and pathological processes [>>21<<,34]. To further explore whether LTA-induced MMP-9 expression mediated through activation of Nox activation, as shown in Figure 2a and b, pretreatment with a Nox activity inhibitor diphenyleneiodonium (DPI, 1 \u03BCM) or p47phox inhibitor" . _:b16174301 . . _:b16174314 . . _:b16174316 . _:b16174278 "Accumulating evidence has shown that MMP-9 is upregulated via an AP-1-dependent manner in various cell types [>>5<<,18]. Upon stimulation, ATF-2 is known to form heterodimers with Jun family transcription factors and bind to a specific AP-1 site in the promoter region of target genes, and enhances the gene transcription [51]. Our data showed that ATF-2" . _:b16174315 . _:b16174299 . _:b16174233 "Moreover, phosphorylation and translocation of p47phox to plasma membrane are important events in the activation of Nox [35,>>36<<]. Therefore, we first determined whether LTA stimulates p47phox phosphorylation at serine residues; as shown in Figure 3a, LTA stimulated p47phox serine phosphorylation in a time-dependent manner, which was attenuated by pretreatment with" . _:b493961931 . . _:b16174310 "Our previous studies have demonstrated that interleukin-1 (IL-1\u03B2), bradykinin (BK) and oxidized low-density lipoprotein (oxLDL) upregulate MMP-9 expression via NF-\u03BAB, Elk-1 and AP-1 signaling in rat astrocytes [4,17,>>18<<]. In response to pathogenic ligands, TLR2/MyD88 activates PI3K/Akt, MAPKs and NF-\u03BAB pathways, which modulate immune responses following ligand recognition [16,19]. Moreover, activation of these signaling cascades and transcription factors" . _:b16174283 . . _:b16174247 . _:b16174254 "Our recent studies have demonstrated that c-Src-dependent NF-\u03BAB and Ca2+/CaMKII-dependent JNK/c-Jun pathways mediate LTA-induced MMP-9 expression and cell migration in RBA-1 cells [>>43<<,44]. Here, we further demonstrated that a PKC\u03B1/Nox2/ROS-dependent ATF2/AP-1 and p300 pathway is also involved in LTA-induced MMP-9 expression and cell migration in brain astrocytes (Figure 7b). Based on these findings, Figure 7c depicts a" . . . _:b16174285 "It was consistent with previous studies indicating p300-dependent MMP-9 expression by BK or IL-1\u03B2 in astrocytes [>>17<<,54] and a recent study showing that ROS-dependent p300 activation leads to cPLA2 expression by cigarette smoke extract in human tracheal smooth muscle cells [55]." . _:b16174272 "Several reports have shown that Nox1, Nox2 and Nox4 are expressed in brain cells [21,>>34<<], suggesting that these Nox isotypes may be essential for ROS generation." . _:b16174320 . _:b16174321 . _:b493961935 . _:b16174228 . _:b16174242 . _:b16174322 . _:b16174251 "Moreover, the gram-positive bacterium Streptococcus pneumoniae is the most common cause of acute bacterial meningitis worldwide [41,>>42<<], revealing a close relationship between LTA challenges and CNS diseases." . _:b493961928 . _:b16174323 . . _:b16174266 . . _:b16174238 . . _:b16174304 "However, the role of MMP-9 in astrocytes, the major regulator of fundamental biological functions of the CNS [>>15<<], in LTA-induced brain inflammation remains poorly defined." . _:b16174294 . . _:b16174293 . . _:b16174243 . _:b16174284 "p300 is vital for the co-activation of several transcription factors such as AP-1 in the transcription machinery, which has a significant role in the activation of AP-1-mediated gene expression for proinflammatory mediators [>>39<<]. Our results indicated that p300 plays an important role in LTA-induced MMP-9 expression (Figure 6a)." . . . _:b16174293 "In brain, MMP-9 has been shown to be upregulated during various CNS diseases [1,>>2<<]. Previous reports have indicated that a series of functional element-binding sites have been identified, including NF-\u03BAB, Ets and AP-1 within the MMP-9 promoter [3], which can be regulated by diverse stimuli. Moreover, proinflammatory" . _:b493961926 . . _:b16174241 . . _:b16174240 . _:b16174323 "Although the effects of LTA on ROS generation have been reported in tracheal smooth muscle cells and renal diseases [26,>>27<<], LTA-induced astrocytic responses through the ROS signal are not well characterized." . _:b16174267 "Consistently, many reports have also shown that ROS are a major signaling factor that mediates microglial activation induced by inflammatory mediators, including LPS [>>46<<]. Herein we are the first group to establish that Nox-dependent ROS generation contributes to upregulation of MMP-9 induced by LTA in brain astrocytes." . _:b16174261 "In the brain, the role of ROS extends to the control of vascular tone, which is tightly modulated by metabolic activity within neurons [21,>>23<<]. Increasing evidence also attributes the cellular damage in neurodegenerative disorders such as AD to oxidative stress that is due to the generation of free radicals implicated in brain inflammatory disorders [24,25,45]. The effects of" . _:b493961917 . . _:b16174238 _:b16174239 . _:b493961934 . . _:b16174294 "Previous reports have indicated that a series of functional element-binding sites have been identified, including NF-\u03BAB, Ets and AP-1 within the MMP-9 promoter [>>3<<], which can be regulated by diverse stimuli." . . _:b16174282 . . . . _:b16174323 . _:b16174256 "The ROS may exert a key role in the normal physiological functions and the inflammatory responses [>>22<<,45]. Several lines of evidence have indicated that ROS play a causative role in numerous disease pathologies such as ischemia/reperfusion injury and degenerative diseases such as atherosclerosis, arthritis and neurodegeneration [24,25]." . _:b16174286 . _:b16174276 "It is consistent with the report indicating that PKC\u03B1 can phosphorylate p47phox, and induce both its translocation and then Nox activation in human neutrophils [>>36<<]." . . _:b16174299 "In the CNS, the glial cells such as astrocytes and microglia are regarded as targets in gram-positive bacterial infection [9,>>10<<]. Lipoteichoic acid (LTA) is a major component of gram-positive bacterial cell walls that induces glial inflammatory activation in vitro and in vivo [11], mediated through TLR2 signaling [12]. In astrocytes, TLR signaling has been shown" . _:b16174228 _:b16174229 . _:b16174228 _:b16174230 . _:b16174228 _:b16174231 . . _:b16174252 . _:b16174228 _:b16174236 . . _:b16174228 _:b16174237 . _:b16174289 "It has been reported that MAPKs, NF-\u03BAB, AP-1 and MMP-9 [>>17<<,18] contribute to cell motility in different cell types." . . _:b16174253 . _:b16174309 . . _:b16174228 _:b16174232 . _:b16174251 . _:b493961924 . _:b16174228 _:b16174233 . . _:b16174228 _:b16174234 . _:b16174228 _:b16174235 . _:b16174314 . . _:b16174256 . . _:b16174296 "It is worth noting that bacterial infections have been found to trigger brain inflammatory diseases [>>7<<]. Gram-positive bacterial infections of the CNS occur in bacterial meningitis and brain abscess, being localized to the membranes surrounding the brain or in its parenchyma, respectively [8]." . _:b16174250 "Moreover, the gram-positive bacterium Streptococcus pneumoniae is the most common cause of acute bacterial meningitis worldwide [>>41<<,42], revealing a close relationship between LTA challenges and CNS diseases." . _:b16174322 "Although the effects of LTA on ROS generation have been reported in tracheal smooth muscle cells and renal diseases [>>26<<,27], LTA-induced astrocytic responses through the ROS signal are not well characterized." . . _:b16174238 _:b16174244 . _:b16174238 _:b16174245 . _:b16174310 . _:b16174238 _:b16174246 . _:b16174277 "Here we focus on the roles of transcription factor AP-1, which is well known to be modulated during oxidative stress associated with inflammatory diseases [>>50<<]. Accumulating evidence has shown that MMP-9 is upregulated via an AP-1-dependent manner in various cell types [5,18]. Upon stimulation, ATF-2 is known to form heterodimers with Jun family transcription factors and bind to a specific AP-1" . _:b16174238 _:b16174240 . _:b16174238 _:b16174241 . _:b16174238 _:b16174242 . _:b16174270 "To date, five Nox isotypes have been discovered, including Nox1-5 [>>34<<]. Several reports have shown that Nox1, Nox2 and Nox4 are expressed in brain cells [21,34], suggesting that these Nox isotypes may be essential for ROS generation." . _:b16174238 _:b16174243 . _:b16174258 "Several lines of evidence have indicated that ROS play a causative role in numerous disease pathologies such as ischemia/reperfusion injury and degenerative diseases such as atherosclerosis, arthritis and neurodegeneration [>>24<<,25]. In the brain, the role of ROS extends to the control of vascular tone, which is tightly modulated by metabolic activity within neurons [21,23]. Increasing evidence also attributes the cellular damage in neurodegenerative disorders" . . . _:b16174254 . _:b16174247 "discussion" . _:b16174321 . _:b16174244 "The peroxide-sensitive fluorescent probe 2\u2032,7\u2032-dichlorofluorescein diacetate (DCF-DA) was used to assess the generation of intracellular ROS [>>31<<] with minor modifications. RBA-1 cells in monolayers were incubated with RPMI-1640 supplemented with 5 \u03BCM DCF-DA for 45 min at 37\u00B0C." . _:b16174320 . . _:b16174289 . . _:b16174320 "Recently, increasing evidence has attributed the cellular damage in neurodegenerative disorders such as AD to oxidative stress, which leads to the generation of free radicals, which are responsible for brain inflammatory disorders [>>24<<,25]. Although the effects of LTA on ROS generation have been reported in tracheal smooth muscle cells and renal diseases [26,27], LTA-induced astrocytic responses through the ROS signal are not well characterized." . . _:b16174272 . . _:b16174319 . _:b16174319 . . _:b16174315 . _:b493961932 . . _:b16174319 . _:b16174235 "Recent reports also suggested that activation of Nox/ROS signaling might be mediated through PKC [>>36<<,38]. Here we further demonstrated which PKC isoforms are involved in LTA-induced Nox/ROS generation and MMP-9 expression. Pretreatment with either a pan-PKC inhibitor GF109203X (30 \u03BCM) or a selective PKC\u03B1 inhibitor G\u00F66976 (1 \u03BCM)" . "10.1186%2F1742-2094-9-110" . _:b16174267 . _:b16174297 . _:b16174312 . _:b16174253 "LTA exhibits detrimental effects on brain cellular functions, including induction of apoptosis, production of oxidative stresses and disruption of BBB following group B Streptococcus or Staphylococcus aureus challenge in the CNS [>>9<<-11]. Despite an obviously important role of LTA in brain diseases, the processes by which LTA was implicated in astrocytic functions are not completely understood." . _:b16174228 . _:b16174245 . _:b16174229 . _:b493961933 . _:b16174230 . . _:b16174290 . _:b16174268 . _:b16174231 . _:b16174271 . _:b16174249 . . . _:b16174232 "Moreover, phosphorylation and translocation of p47phox to plasma membrane are important events in the activation of Nox [>>35<<,36]. Therefore, we first determined whether LTA stimulates p47phox phosphorylation at serine residues; as shown in Figure 3a, LTA stimulated p47phox serine phosphorylation in a time-dependent manner, which was attenuated by pretreatment" . _:b16174279 "Accumulating evidence has shown that MMP-9 is upregulated via an AP-1-dependent manner in various cell types [5,>>18<<]. Upon stimulation, ATF-2 is known to form heterodimers with Jun family transcription factors and bind to a specific AP-1 site in the promoter region of target genes, and enhances the gene transcription [51]. Our data showed that ATF-2" . _:b16174236 . _:b16174257 . _:b16174237 . _:b16174241 "Total RNA was extracted from RBA-1 cells [>>29<<]. The cDNA obtained from 0.5 \u03BCg total RNA was used as a template for PCR amplification." . _:b16174247 _:b16174288 . _:b16174247 _:b16174289 . _:b16174238 . _:b16174247 _:b16174290 . _:b16174260 . _:b16174239 . _:b16174282 "Moreover, AP-1 binding sites have been identified in the MMP-9 gene promoter [>>3<<], which might explain the modulation exerted by LTA." . _:b16174239 "Primary astrocyte cultures were prepared from the cortex of 6-day-old Sprague\u2013Dawley rat pups [>>29<<]. Rats were maintained according to the Guidelines of the Animal Care Committee of Chang Gung University and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The purity of primary astrocyte cultures was" . _:b16174255 "Our recent studies have demonstrated that c-Src-dependent NF-\u03BAB and Ca2+/CaMKII-dependent JNK/c-Jun pathways mediate LTA-induced MMP-9 expression and cell migration in RBA-1 cells [43,>>44<<]. Here, we further demonstrated that a PKC\u03B1/Nox2/ROS-dependent ATF2/AP-1 and p300 pathway is also involved in LTA-induced MMP-9 expression and cell migration in brain astrocytes (Figure 7b). Based on these findings, Figure 7c depicts a" . _:b16174247 _:b16174280 . _:b16174232 . _:b16174247 _:b16174281 . _:b16174305 . _:b16174247 _:b16174282 . _:b16174302 "In astrocytes, TLR signaling has been shown to be involved in brain inflammatory responses [>>13<<], accompanied by upregulation of several genes with proinflammatory and proapoptotic capabilities [14]." . _:b16174247 _:b16174283 . _:b16174247 _:b16174284 . _:b16174233 . _:b16174247 _:b16174285 . _:b16174279 . _:b16174247 _:b16174286 . _:b16174247 _:b16174287 . _:b16174247 _:b16174272 . _:b16174234 . _:b16174247 _:b16174273 . _:b16174244 . _:b16174247 _:b16174274 . _:b16174247 _:b16174275 . _:b16174247 _:b16174276 . _:b16174303 "In astrocytes, TLR signaling has been shown to be involved in brain inflammatory responses [13], accompanied by upregulation of several genes with proinflammatory and proapoptotic capabilities [>>14<<]. However, the role of MMP-9 in astrocytes, the major regulator of fundamental biological functions of the CNS [15], in LTA-induced brain inflammation remains poorly defined." . _:b16174276 . _:b16174264 . _:b16174235 . _:b16174247 _:b16174277 . _:b16174247 _:b16174278 . _:b16174247 _:b16174279 . _:b493961919 . _:b16174247 _:b16174264 . _:b16174273 "These results are consistent with previous studies showing that Nox is expressed in astrocytes, contributes to ROS generation [>>47<<,48] and leads to LPS-induced MMPs expression in Raw264.7 cells [49]." . _:b16174244 . _:b16174247 _:b16174265 . _:b16174292 . _:b16174247 _:b16174266 . _:b16174308 "Our previous studies have demonstrated that interleukin-1 (IL-1\u03B2), bradykinin (BK) and oxidized low-density lipoprotein (oxLDL) upregulate MMP-9 expression via NF-\u03BAB, Elk-1 and AP-1 signaling in rat astrocytes [>>4<<,17,18]. In response to pathogenic ligands, TLR2/MyD88 activates PI3K/Akt, MAPKs and NF-\u03BAB pathways, which modulate immune responses following ligand recognition [16,19]. Moreover, activation of these signaling cascades and transcription" . _:b16174255 . _:b16174247 _:b16174267 . _:b16174275 "These results are consistent with previous studies showing that Nox is expressed in astrocytes, contributes to ROS generation [47,48] and leads to LPS-induced MMPs expression in Raw264.7 cells [>>49<<]. We also suggested that the LTA-stimulated p47phox-dependent Nox2/ROS signal and MMP-9 expression might be mediated through PKC\u03B1 in RBA-1 cells (Figure 4). It is consistent with the report indicating that PKC\u03B1 can phosphorylate p47phox," . _:b16174247 _:b16174268 . _:b16174295 . _:b16174245 . _:b16174247 _:b16174269 . "PMC0" . . _:b16174247 _:b16174270 . _:b16174247 _:b16174271 . _:b16174234 . _:b16174231 "NADPH oxidase (Nox) is considered to be a major source of ROS in several physiological and pathological processes [21,>>34<<]. To further explore whether LTA-induced MMP-9 expression mediated through activation of Nox activation, as shown in Figure 2a and b, pretreatment with a Nox activity inhibitor diphenyleneiodonium (DPI, 1 \u03BCM) or p47phox inhibitor apocynin" . _:b16174247 _:b16174256 . _:b16174246 . _:b16174247 _:b16174257 . _:b16174311 . _:b16174304 . _:b16174247 _:b16174258 . _:b16174247 _:b16174259 . _:b16174285 . _:b16174247 _:b16174260 . _:b16174307 . _:b16174247 _:b16174261 . _:b16174313 "Moreover, activation of these signaling cascades and transcription factors has been reported to be involved in induction of MMP-9 in rat astrocytes [>>4<<,17,18]. A recent report has shown that LTA increases MMP-9 expression via the ERK pathway in RAW 264.7 macrophages [20]. However, the mechanisms underlying the regulation of MMP-9 expression by LTA in astrocytes are still unclear." . _:b16174247 . _:b16174236 . _:b16174247 _:b16174262 . _:b16174247 _:b16174263 . _:b16174247 _:b16174248 . _:b16174296 . _:b16174288 . _:b16174240 . _:b16174247 _:b16174249 . _:b16174247 _:b16174250 . . _:b16174247 _:b16174251 . _:b16174280 "Upon stimulation, ATF-2 is known to form heterodimers with Jun family transcription factors and bind to a specific AP-1 site in the promoter region of target genes, and enhances the gene transcription [>>51<<]. Our data showed that ATF-2 phosphorylation is essential for LTA-induced MMP-9 expression, determined by AP-1 inhibitor (TSIIA) (Figure 5). Such inhibitory effects were also confirmed by transfection with ATF-2 siRNA on MMP-9 expression" . _:b16174247 _:b16174252 . _:b16174241 . _:b16174247 _:b16174253 . _:b16174247 _:b16174254 . _:b16174247 _:b16174255 . _:b16174242 . _:b16174295 . _:b16174243 . _:b16174314 "Moreover, activation of these signaling cascades and transcription factors has been reported to be involved in induction of MMP-9 in rat astrocytes [4,>>17<<,18]. A recent report has shown that LTA increases MMP-9 expression via the ERK pathway in RAW 264.7 macrophages [20]. However, the mechanisms underlying the regulation of MMP-9 expression by LTA in astrocytes are still unclear." . _:b16174252 . _:b16174229 . _:b16174253 . _:b16174262 "Increasing evidence also attributes the cellular damage in neurodegenerative disorders such as AD to oxidative stress that is due to the generation of free radicals implicated in brain inflammatory disorders [>>24<<,25,45]. The effects of LTA on ROS generation have been reported in human renal [27] and tracheal diseases [26]. In this study, we found and confirmed that LTA-induced MMP-9 expression is mediated through Nox-dependent ROS generation in" . _:b16174254 . . _:b493961930 . . _:b16174255 . . _:b16174238 "methods" . _:b16174298 . _:b16174248 . _:b16174264 "Increasing evidence also attributes the cellular damage in neurodegenerative disorders such as AD to oxidative stress that is due to the generation of free radicals implicated in brain inflammatory disorders [24,25,>>45<<]. The effects of LTA on ROS generation have been reported in human renal [27] and tracheal diseases [26]. In this study, we found and confirmed that LTA-induced MMP-9 expression is mediated through Nox-dependent ROS generation in RBA-1" . _:b16174237 "Several studies have reported that p300, a transcriptional co-activator, plays a critical role in many gene transcription-regulating events [>>39<<]. Hence, we also investigated the role of p300 in LTA-induced MMP-9 expression. Pretreatment with the p300 inhibitor garcinol (GR343, 0.1-10 \u03BCM) concentration-dependently inhibited LTA-induced MMP-9 expression (Figure 6a)." . _:b16174249 . . _:b16174250 . . _:b16174251 . _:b16174322 . _:b16174260 . _:b16174261 . _:b16174281 "Theses results are consistent with the mechanisms of MMP-2 expression in human breast epithelial cells [>>52<<]. Moreover, AP-1 binding sites have been identified in the MMP-9 gene promoter [3], which might explain the modulation exerted by LTA. Our data further showed that LTA enhanced phospho-ATF-2 binding to MMP-9 promoter, determined by a" . _:b16174240 "The cultured media were analyzed by gelatin zymography [>>29<<]. Gelatinolytic activity was manifested as horizontal white bands on a blue background." . _:b16174262 . _:b16174277 . . _:b16174259 "Several lines of evidence have indicated that ROS play a causative role in numerous disease pathologies such as ischemia/reperfusion injury and degenerative diseases such as atherosclerosis, arthritis and neurodegeneration [24,>>25<<]. In the brain, the role of ROS extends to the control of vascular tone, which is tightly modulated by metabolic activity within neurons [21,23]. Increasing evidence also attributes the cellular damage in neurodegenerative disorders such" . _:b16174300 . _:b493961917 "2"^^ . _:b16174308 . _:b16174263 . . _:b16174256 . _:b493961920 . _:b493961919 "2"^^ . _:b16174257 . _:b16174291 "background" . . _:b16174258 . _:b493961918 "2"^^ . _:b16174260 "In the brain, the role of ROS extends to the control of vascular tone, which is tightly modulated by metabolic activity within neurons [>>21<<,23]. Increasing evidence also attributes the cellular damage in neurodegenerative disorders such as AD to oxidative stress that is due to the generation of free radicals implicated in brain inflammatory disorders [24,25,45]. The effects" . . . . _:b16174259 . _:b16174268 . _:b16174269 . _:b16174307 "Ultimately, TLR signaling activates the MAPK family, NF-\u03BAB and AP-1, leading to expression of cytokines and other proinflammatory proteins [7,>>8<<]. Our previous studies have demonstrated that interleukin-1 (IL-1\u03B2), bradykinin (BK) and oxidized low-density lipoprotein (oxLDL) upregulate MMP-9 expression via NF-\u03BAB, Elk-1 and AP-1 signaling in rat astrocytes [4,17,18]. In response to" . . _:b16174257 "The ROS may exert a key role in the normal physiological functions and the inflammatory responses [22,>>45<<]. Several lines of evidence have indicated that ROS play a causative role in numerous disease pathologies such as ischemia/reperfusion injury and degenerative diseases such as atherosclerosis, arthritis and neurodegeneration [24,25]." . _:b16174270 . . . _:b493961925 "2"^^ . _:b16174321 "Recently, increasing evidence has attributed the cellular damage in neurodegenerative disorders such as AD to oxidative stress, which leads to the generation of free radicals, which are responsible for brain inflammatory disorders [24,>>25<<]. Although the effects of LTA on ROS generation have been reported in tracheal smooth muscle cells and renal diseases [26,27], LTA-induced astrocytic responses through the ROS signal are not well characterized." . _:b16174271 . _:b493961924 "2"^^ . _:b16174284 . _:b16174264 . _:b16174245 "The NADPH oxidase activity in intact cells was assayed by lucigenin chemiluminescence assay as described previously [>>32<<] with modifications. After incubation, the cells were gently scraped and centrifuged at 400\u2009\u00D7\u2009g for 10 min at 4\u00B0C." . _:b16174230 . _:b493961927 "2"^^ . _:b16174265 . _:b16174236 "Recent reports also suggested that activation of Nox/ROS signaling might be mediated through PKC [36,>>38<<]. Here we further demonstrated which PKC isoforms are involved in LTA-induced Nox/ROS generation and MMP-9 expression. Pretreatment with either a pan-PKC inhibitor GF109203X (30 \u03BCM) or a selective PKC\u03B1 inhibitor G\u00F66976 (1 \u03BCM)" . _:b16174318 "ROS are essential for many physiological functions and killing invading microorganisms [21,>>22<<]. Under pathological conditions, increasing ROS production by external stimuli can regulate the expression of several inflammatory genes during brain injury [23-25]. Recently, increasing evidence has attributed the cellular damage in" . _:b16174229 "ROS have been shown to induce MMPs expression in various cell types [>>33<<]. To determine whether ROS participated in MMP-9 induction, pretreatment of RBA-1 cells with N-acetylcysteine (NAC, a ROS scavenger, 10 mM) attenuated the LTA-induced MMP-9 protein and mRNA expression (Figure 1a and b), suggesting that" . _:b493961926 "2"^^ . _:b16174306 . _:b16174266 . . _:b493961921 "2"^^ . _:b16174267 . . _:b16174263 . _:b16174291 _:b16174312 . _:b16174313 . _:b16174276 . _:b493961920 "2"^^ . _:b16174291 _:b16174313 . _:b16174291 _:b16174314 . _:b16174239 . _:b16174262 . _:b16174291 _:b16174315 . _:b16174291 _:b16174316 . _:b16174277 . _:b493961923 "2"^^ . _:b16174291 _:b16174317 . _:b16174291 _:b16174318 . _:b16174243 "The phosphorylation of serine, ATF2, and p300 or total Nox, p47phox, PKC\u03B1, ATF2 and p300 were identified and quantified by Western blotting as previously described [>>29<<]. The immunoreactive bands were detected by the UVP Biospectrum\u00AE imaging system (Upland, CA)." . _:b16174291 _:b16174319 . _:b16174291 _:b16174304 . _:b16174278 . _:b16174261 . _:b493961922 "2"^^ . _:b16174291 _:b16174305 . . _:b16174291 _:b16174306 . _:b16174266 "The effects of LTA on ROS generation have been reported in human renal [27] and tracheal diseases [>>26<<]. In this study, we found and confirmed that LTA-induced MMP-9 expression is mediated through Nox-dependent ROS generation in RBA-1 cells (Figure 1). Consistently, many reports have also shown that ROS are a major signaling factor that" . _:b16174291 _:b16174307 . _:b16174291 _:b16174308 . _:b16174279 . _:b493961933 "2"^^ . _:b16174291 _:b16174309 . _:b16174291 _:b16174310 . _:b16174291 _:b16174311 . _:b16174319 "Under pathological conditions, increasing ROS production by external stimuli can regulate the expression of several inflammatory genes during brain injury [>>23<<-25]. Recently, increasing evidence has attributed the cellular damage in neurodegenerative disorders such as AD to oxidative stress, which leads to the generation of free radicals, which are responsible for brain inflammatory disorders" . _:b16174259 . . _:b16174272 . _:b493961932 "2"^^ . . _:b16174258 . _:b493961935 "2"^^ . _:b16174290 "It has been reported that MAPKs, NF-\u03BAB, AP-1 and MMP-9 [17,>>18<<] contribute to cell motility in different cell types." . _:b16174273 . _:b16174283 "These results suggested that LTA stimulated ATF-2/AP-1 phosphorylation and binding activity via a ROS-dependent pathway, consistent with ROS-dependent activation of ATF-2 being involved in H9c2 cells [>>53<<]." . _:b16174291 _:b16174320 . _:b16174274 . _:b493961934 "2"^^ . _:b16174291 _:b16174321 . _:b16174291 _:b16174322 . _:b16174291 _:b16174323 . _:b493961929 "2"^^ . _:b16174275 . . _:b493961928 "2"^^ . _:b16174284 . _:b16174269 . _:b16174292 "In brain, MMP-9 has been shown to be upregulated during various CNS diseases [>>1<<,2]. Previous reports have indicated that a series of functional element-binding sites have been identified, including NF-\u03BAB, Ets and AP-1 within the MMP-9 promoter [3], which can be regulated by diverse stimuli. Moreover, proinflammatory" . _:b493961931 "2"^^ . _:b16174285 . _:b493961927 . . _:b16174286 "It was consistent with previous studies indicating p300-dependent MMP-9 expression by BK or IL-1\u03B2 in astrocytes [17,>>54<<] and a recent study showing that ROS-dependent p300 activation leads to cPLA2 expression by cigarette smoke extract in human tracheal smooth muscle cells [55]." . _:b493961930 "2"^^ . _:b16174286 . _:b16174246 . _:b16174295 . _:b16174288 "Cell motility is a fundamental process during embryonic development, wound healing, inflammatory responses and tumor metastasis [>>56<<]. It has been reported that MAPKs, NF-\u03BAB, AP-1 and MMP-9 [17,18] contribute to cell motility in different cell types." . _:b16174287 . . _:b16174300 "Lipoteichoic acid (LTA) is a major component of gram-positive bacterial cell walls that induces glial inflammatory activation in vitro and in vivo [>>11<<], mediated through TLR2 signaling [12]." . _:b16174248 "Blockade of MMP-9 activity by pharmacological inhibitors or gene knockout strategies provide protective effects against cerebral ischemia [>>39<<,40]. Moreover, the gram-positive bacterium Streptococcus pneumoniae is the most common cause of acute bacterial meningitis worldwide [41,42], revealing a close relationship between LTA challenges and CNS diseases. The pathogenic" . _:b16174280 . . _:b16174291 _:b16174296 . _:b16174280 . . _:b16174291 _:b16174297 . _:b493961917 . . _:b16174291 _:b16174298 . _:b16174291 _:b16174299 . _:b16174291 _:b16174300 . _:b16174281 . _:b16174265 "The effects of LTA on ROS generation have been reported in human renal [>>27<<] and tracheal diseases [26]." . . _:b16174291 _:b16174301 . _:b16174291 _:b16174302 . . _:b16174291 _:b16174303 . _:b16174282 . . _:b493961919 . _:b16174302 . _:b16174291 _:b16174292 . _:b16174283 . _:b16174246 "To detect the in vivo association of nuclear proteins with rat mmp-9 promoter, chromatin immunoprecipitation (ChIP) analysis was conducted as described previously [>>17<<]. RBA-1 cells in 100-mm dishes were grown to confluence and serum starved for 24 h." . _:b16174291 _:b16174293 . _:b493961918 . _:b16174291 _:b16174294 . _:b16174291 _:b16174295 . . _:b16174292 . _:b493961936 "2"^^ . _:b16174270 . _:b16174305 "TLR2 is believed to be responsible for LTA recognition challenged by gram-positive bacteria such as Staphylococcus aureus and Streptococcus pneumouniae[>>16<<]. Upon binding to TLR heterodimers (i.e., TLR2/TLR1 or TLR2/TLR6 complex), LTA exerts a sequential activation of members of IL-1 receptor-associated kinase (IRAK) family and tumor necrosis factor receptor-associated factor 6 (TRAF6)," . _:b16174263 "Increasing evidence also attributes the cellular damage in neurodegenerative disorders such as AD to oxidative stress that is due to the generation of free radicals implicated in brain inflammatory disorders [24,>>25<<,45]. The effects of LTA on ROS generation have been reported in human renal [27] and tracheal diseases [26]. In this study, we found and confirmed that LTA-induced MMP-9 expression is mediated through Nox-dependent ROS generation in RBA-1" . _:b16174287 "indicating p300-dependent MMP-9 expression by BK or IL-1\u03B2 in astrocytes [17,54] and a recent study showing that ROS-dependent p300 activation leads to cPLA2 expression by cigarette smoke extract in human tracheal smooth muscle cells [>>55<<]. Subsequently, we also demonstrated that LTA-stimulated MMP-9 transcriptional activity via the same pathway by a wild-type rat MMP-9 promoter-luciferase reporter plasmid (pGL-MMP-9-Luc) construct (Figure 7a)." . _:b16174265 . _:b16174293 . _:b16174252 . _:b16174294 . . _:b16174253 . _:b16174317 "ROS are essential for many physiological functions and killing invading microorganisms [>>21<<,22]. Under pathological conditions, increasing ROS production by external stimuli can regulate the expression of several inflammatory genes during brain injury [23-25]. Recently, increasing evidence has attributed the cellular damage in" . _:b16174295 . . _:b493961917 . _:b16174288 . _:b493961918 . _:b16174253 . _:b493961919 . _:b493961925 . _:b16174289 . _:b16174252 . _:b493961924 . _:b16174290 . _:b493961927 . _:b16174275 . . _:b16174291 . _:b493961926 . _:b16174301 "Lipoteichoic acid (LTA) is a major component of gram-positive bacterial cell walls that induces glial inflammatory activation in vitro and in vivo [11], mediated through TLR2 signaling [>>12<<]. In astrocytes, TLR signaling has been shown to be involved in brain inflammatory responses [13], accompanied by upregulation of several genes with proinflammatory and proapoptotic capabilities [14]. However, the role of MMP-9 in" . _:b16174248 . _:b16174300 . _:b493961921 . _:b16174269 "The Nox is considered to be a major source of ROS in several physiological and pathological processes [21,>>35<<]. To date, five Nox isotypes have been discovered, including Nox1-5 [34]. Several reports have shown that Nox1, Nox2 and Nox4 are expressed in brain cells [21,34], suggesting that these Nox isotypes may be essential for ROS generation." . _:b16174301 . _:b493961920 . . _:b16174302 . _:b493961923 . _:b16174298 "In the CNS, the glial cells such as astrocytes and microglia are regarded as targets in gram-positive bacterial infection [>>9<<,10]. Lipoteichoic acid (LTA) is a major component of gram-positive bacterial cell walls that induces glial inflammatory activation in vitro and in vivo [11], mediated through TLR2 signaling [12]. In astrocytes, TLR signaling has been" . _:b16174242 "Oligonucleotide primers were designed based on Genbank entries for rat MMP-9, Nox1-4 [>>30<<] and \u03B2-actin." . _:b16174303 . _:b16174228 "results" . _:b493961922 . _:b16174278 . _:b493961936 . _:b16174296 . . _:b493961933 . _:b16174297 . _:b493961918 . _:b493961932 . _:b16174295 "Moreover, proinflammatory factors including cytokines, endotoxins and oxidative stress have been reported to upregulate MMP-9 in astrocytes in vitro [>>4<<-6], implying that MMP-9 activity may be regulated by diverse factors in the CNS during neuroinflammation." . _:b16174298 . _:b16174234 "Several PKC isoforms such as PKC-\u03B1 have been shown to regulate MMP-9 expression in various cell types [>>37<<]. Recent reports also suggested that activation of Nox/ROS signaling might be mediated through PKC [36,38]." . _:b493961935 . _:b16174297 "Gram-positive bacterial infections of the CNS occur in bacterial meningitis and brain abscess, being localized to the membranes surrounding the brain or in its parenchyma, respectively [>>8<<]. In the CNS, the glial cells such as astrocytes and microglia are regarded as targets in gram-positive bacterial infection [9,10]. Lipoteichoic acid (LTA) is a major component of gram-positive bacterial cell walls that induces glial" . _:b16174299 . _:b493961934 . . . _:b16174308 . _:b16174274 "These results are consistent with previous studies showing that Nox is expressed in astrocytes, contributes to ROS generation [47,>>48<<] and leads to LPS-induced MMPs expression in Raw264.7 cells [49]." . _:b493961929 . _:b16174268 "The Nox is considered to be a major source of ROS in several physiological and pathological processes [>>21<<,35]. To date, five Nox isotypes have been discovered, including Nox1-5 [34]. Several reports have shown that Nox1, Nox2 and Nox4 are expressed in brain cells [21,34], suggesting that these Nox isotypes may be essential for ROS generation." . _:b16174309 . _:b16174271 "Several reports have shown that Nox1, Nox2 and Nox4 are expressed in brain cells [>>21<<,34], suggesting that these Nox isotypes may be essential for ROS generation." . _:b493961928 . . _:b16174317 . _:b16174287 . _:b16174310 . _:b493961931 . _:b16174311 . _:b493961930 . _:b16174304 . _:b493961922 . _:b16174233 . _:b493961921 . _:b16174305 . _:b16174315 "Moreover, activation of these signaling cascades and transcription factors has been reported to be involved in induction of MMP-9 in rat astrocytes [4,17,>>18<<]. A recent report has shown that LTA increases MMP-9 expression via the ERK pathway in RAW 264.7 macrophages [20]. However, the mechanisms underlying the regulation of MMP-9 expression by LTA in astrocytes are still unclear." . _:b16174250 . _:b16174303 . _:b493961929 . _:b493961924 . _:b493961925 . _:b16174306 . _:b493961926 . _:b493961927 . . _:b493961920 . _:b16174235 . _:b493961921 . _:b16174312 "In response to pathogenic ligands, TLR2/MyD88 activates PI3K/Akt, MAPKs and NF-\u03BAB pathways, which modulate immune responses following ligand recognition [16,>>19<<]. Moreover, activation of these signaling cascades and transcription factors has been reported to be involved in induction of MMP-9 in rat astrocytes [4,17,18]. A recent report has shown that LTA increases MMP-9 expression via the ERK" . _:b16174307 . _:b16174306 "Ultimately, TLR signaling activates the MAPK family, NF-\u03BAB and AP-1, leading to expression of cytokines and other proinflammatory proteins [>>7<<,8]. Our previous studies have demonstrated that interleukin-1 (IL-1\u03B2), bradykinin (BK) and oxidized low-density lipoprotein (oxLDL) upregulate MMP-9 expression via NF-\u03BAB, Elk-1 and AP-1 signaling in rat astrocytes [4,17,18]. In response" . _:b16174273 . _:b16174231 . _:b493961922 . _:b16174291 . _:b16174309 "Our previous studies have demonstrated that interleukin-1 (IL-1\u03B2), bradykinin (BK) and oxidized low-density lipoprotein (oxLDL) upregulate MMP-9 expression via NF-\u03BAB, Elk-1 and AP-1 signaling in rat astrocytes [4,>>17<<,18]. In response to pathogenic ligands, TLR2/MyD88 activates PI3K/Akt, MAPKs and NF-\u03BAB pathways, which modulate immune responses following ligand recognition [16,19]. Moreover, activation of these signaling cascades and transcription" . _:b493961923 . _:b493961932 . _:b493961933 . _:b16174316 "A recent report has shown that LTA increases MMP-9 expression via the ERK pathway in RAW 264.7 macrophages [>>20<<]. However, the mechanisms underlying the regulation of MMP-9 expression by LTA in astrocytes are still unclear." . _:b16174316 . _:b493961934 . _:b493961935 . . _:b493961928 . _:b493961929 . _:b16174317 . _:b493961930 . _:b16174318 . _:b493961936 . _:b493961931 . _:b493961923 . _:b16174318 . _:b16174249 "Blockade of MMP-9 activity by pharmacological inhibitors or gene knockout strategies provide protective effects against cerebral ischemia [39,>>40<<]. Moreover, the gram-positive bacterium Streptococcus pneumoniae is the most common cause of acute bacterial meningitis worldwide [41,42], revealing a close relationship between LTA challenges and CNS diseases. The pathogenic progression" . _:b16174274 . _:b493961936 . _:b16174237 . _:b16174319 . _:b16174232 . _:b16174311 "In response to pathogenic ligands, TLR2/MyD88 activates PI3K/Akt, MAPKs and NF-\u03BAB pathways, which modulate immune responses following ligand recognition [>>16<<,19]. Moreover, activation of these signaling cascades and transcription factors has been reported to be involved in induction of MMP-9 in rat astrocytes [4,17,18]. A recent report has shown that LTA increases MMP-9 expression via the ERK" . _:b16174312 . _:b16174281 .