NADPH oxidase-mediated redox signal contributes to lipoteichoic acid-induced MMP-9 upregulation in brain astrocytes

Abstract

Background: Lipoteichoic acid (LTA) is a component of gram-positive bacterial cell walls and may be elevated in the cerebrospinal fluid of patients suffering from meningitis. Among matrix metalloproteinases (MMPs), MMP-9 has been observed in patients with brain inflammatory diseases and may contribute to the pathology of brain diseases. Moreover, several studies have suggested that increased oxidative stress is implicated in the pathogenesis of brain inflammation and injury. However, the molecular mechanisms underlying LTA-induced redox signal and MMP-9 expression in brain astrocytes remain unclear.

Objective: Herein we explored whether LTA-induced MMP-9 expression was mediated through redox signals in rat brain astrocytes (RBA-1 cells).

Methods: Upregulation of MMP-9 by LTA was evaluated by zymographic and RT-PCR analyses. Next, the MMP-9 regulatory pathways were investigated by pretreatment with pharmacological inhibitors or transfection with small interfering RNAs (siRNAs), Western blotting, and chromatin immunoprecipitation (ChIP)-PCR and promoter activity reporter assays. Moreover, we determined the cell functional changes by migration assay.

Results: These results showed that LTA induced MMP-9 expression via a PKC(α)-dependent pathway. We further demonstrated that PKCα stimulated p47phox/NADPH oxidase 2 (Nox2)-dependent reactive oxygen species (ROS) generation and then activated the ATF2/AP-1 signals. The activated-ATF2 bound to the AP-1-binding site of MMP-9 promoter, and thereby turned on MMP-9 gene transcription. Additionally, the co-activator p300 also contributed to these responses. Functionally, LTA-induced MMP-9 expression enhanced astrocytic migration.

Conclusion: These results demonstrated that in RBA-1 cells, activation of ATF2/AP-1 by the PKC(α)-mediated Nox(2)/ROS signals is essential for upregulation of MMP-9 and cell migration enhanced by LTA.

Figure 1

ROS are essential for LTA-induced MMP-9 expression in RBA-1 cells. a Cells were pretreated with or without N-acetylcysteine (NAC, 10 mM) for 1 h before exposure to 50 μg/ml LTA for the indicated time intervals. The conditioned media were analyzed by gelatin zymography for MMP-9 expression and activity. b Cells were treated with or without NAC (10 mM) for 1 h before exposure to 50 μg/ml LTA for 16 h. The total RNA was analyzed by RT-PCR (upper panel) and real-time RT-PCR (lower panel). c Cells were incubated with the peroxide-sensitive fluorescent probe DCF-DA (5 μM) for 45 min, followed by stimulation with 50 μg/ml LTA for the indicated time intervals or for 15 min in presence or absence of 10 mM NAC. The fluorescence intensity of cells was determined. d Rat primary astrocytes were isolated and cultured, and pretreated with or without NAC (1 or 10 mM) before exposure to LTA for 16 h. The conditioned media were analyzed by gelatin zymography (upper panel). For cell migration, cells were plated on coverslips and grown to confluence, then the coverslips were transferred to a new 10-cm dish containing serum-free medium for 24 h. Cells were pretreated with MMP2/9 inhibitor (2/9i) or NAC for 1 h and then incubated with LTA (50 μg/ml) for 48 h. Phase contrast images of cells were taken. The number of LTA-induced cell migrations was counted (lower panel). Data are expressed as the mean ± SEM of three independent experiments. ^*P < 0.05; ^#P < 0.01, as compared with the respective values of cells stimulated with vehicle (c) or LTA alone (c, d). The figure represents one of three similar experiments.

Figure 2

NADPH oxidase (Nox)-dependent ROS generation is involved in LTA-induced MMP-9 expression in RBA-1 cells. a Cells were pretreated with or without DPI (1 μM) or apocynin (Apo, 10 μM) for 1 h before exposure to LTA (50 μg/ml) for the indicated time intervals. The conditioned media were analyzed by gelatin zymography for MMP-9 expression and activity. b The total RNA was collected and MMP-9 mRNA expression analyzed by RT-PCR (upper panel) and real-time RT-PCR (lower panel). c Cells were pretreated with or without DPI (1 μM) or apocynin (Apo, 10 μM) for 1 h before exposure to 50 μg/ml LTA for 15 min. The Nox activity and ROS generation were analyzed. d Cells were transfected with scramble (scra) or Nox2 siRNA for 24 h, followed by stimulation with LTA for 16 h. The conditioned media and cell lysates were collected for gelatin zymography or Western blotting analysis of Nox2 and GAPDH (as an internal control). Data are expressed as the mean ± SEM of three independent experiments. ^*P < 0.05; ^#P < 0.01, as compared with the r cells stimulated with LTA (c) alone. The figure represents one of three similar experiments.

Figure 3

Involvement of p47^phoxtranslocation in LTA-induced MMP-9 expression. Cells were pretreated without or with Apo (10 μM) for 1 h and then stimulated with 50 μg/ml LTA for the indicated time intervals or 10 min. a The whole cell lysates and (b) the membrane fraction were prepared and analyzed by Western blotting using an anti-phospho-serine (p-Ser), p47^phox or Gαs (as a membrane control) antibody. The p47^phox translocation was also observed by immunofluorescent staining. c Cells were transfected with scramble (scra) or p47^phox siRNA for 24 h, followed by stimulation with LTA for 16 h. The conditioned media and cell lysates were analyzed by gelatin zymography or Western blotting analysis of p47^phox and GAPDH. Data are expressed as the mean of three independent experiments. ^*P < 0.05; ^#P < 0.01, as compared with the cells stimulated with vehicle (a) and LTA (a, b) alone. ^§P < 0.01, as compared with the cells stimulated with LTA (a) alone. The figure represents one of three similar experiments.

Figure 4

LTA induces ROS generation and MMP-9 expression via PKCα. a Cells were pretreated with or without GF109203X (GF, 30 μM) or Gö6976 (Gö, 1 μM) for 1 h before exposure to 50 μg/ml LTA for the indicated time intervals. The conditioned media were analyzed by gelatin zymography. b Cells were pretreated without or with Gö (1 μM) for 1 h and then incubated with LTA for the indicated time intervals (upper part) or 10 min (lower part). The membrane and cytosol fractions were prepared and analyzed by Western blotting using anti-PKC-α, Gαs (as a membrane control) or GAPDH antibody. c Cells were pretreated with or without GF or Gö for 1 h before exposure to LTA for 15 min. The Nox activity and ROS generation were analyzed. d Cells were pretreated with or without Gö (1 μM) for 1 h before exposure to LTA for the indicated time intervals. e Cells were transfected with scramble (scra) or PKCα siRNA for 24 h, followed by stimulation with LTA for 16 h. The conditioned media and cell lysates were analyzed by gelatin zymography or Western blotting analysis. The cell lysates were analyzed by Western blotting using anti-phospho-serine (p-Ser), p47^phox or GAPDH antibody. Data are expressed as the mean ± SEM of three independent experiments. ^*P < 0.01; ^#P < 0.01, as compared with the cells stimulated with LTA (c, d) alone. The figure represents one of three similar experiments.

Figure 5

ATF-2/AP-1 is required for LTA-induced MMP-9 expression. a RBA-1 cells were transfected with scramble (scra) or ATF2 siRNA for 24 h, followed by stimulation with 50 μg/ml LTA for 16 h. The conditioned media and cell lysates were analyzed by gelatin zymography or Western blotting. b Cells were treated with LTA for the indicated time intervals. c Cells were pretreated with or without tanshinone IIA (TSIIA, 10 μM), Gö6976 (1 μM), NAC (10 mM), DPI (1 μM) or apocynin (10 μM) for 1 h before exposure to LTA for the indicated time intervals. The cell lysates were analyzed by Western blotting using an anti-phospho-ATF-2 (p-ATF2) or GAPDH antibody. d RBA-1 cells were pretreated with NAC (10 mM) for 1 h and then incubated with LTA (50 μg/ml) for the indicated time intervals or 90 min. The ATF-2/AP-1 binding activity was analyzed by chromatin-IP (ChIP)-PCR assay. Data are expressed as the mean of three independent experiments. ^#P < 0.01, as compared with the cells stimulated with vehicle (b, d) or ^§P < 0.05, as compared with the cells stimulated with LTA (d) alone. The figure represents one of three similar experiments.

Figure 6

The transcriptional co-activator p300 contributes to LTA-induced MMP-9 expression. a Cells were pretreated with or without GR343 for 1 h before exposure to 50 μg/ml LTA for 16 h. The conditioned media were analyzed by gelatin zymography. b, c Cells were pretreated without or with Gö6976 (Gö, 1 μM), DPI (1 μM), apocynin (10 μM) or NAC (10 mM) for 1 h, and then incubated with 50 μg/ml LTA for the indicated time intervals (b) or 90 min (c). The cell lysates were analyzed by Western blotting using an anti-phospho-p300 (p-p300) or GAPDH antibody. d Cells were transfected with scramble (scra) or p300 siRNA for 24 h, followed by stimulation with 50 μg/ml LTA for 16 h. The conditioned media and cell lysates were collected and analyzed by gelatin zymography or Western blotting. Data are expressed as the mean of three independent experiments. ^*P < 0.05; ^#P < 0.01, as compared with the cells stimulated with vehicle (b) or LTA (a, c) alone. The figure represents one of three similar experiments.

Figure 7

LTA enhances MMP-9 promoter activity and astrocytic migration. a Cells were transiently cotransfected with pGL-MMP9-Luc and pGal for 24 h, pretreated with Gö, DPI, Apo, NAC and GR343 for 1 h, and then incubated with LTA for 16 h. The promoter activity was determined. b RBA-1 cells were plated on coverslips and grew to confluence; the coverslips were transferred to a new 10-cm dish containing serum-free medium for 24 h. Cells were pretreated with MMP2/9i, Gö, DPI, NAC or GR343 for 1 h, and then incubated with LTA (50 μg/ml) for 48 h. Phase contrast images of RBA-1 cells were taken. The number of LTA-induced cell migrations was counted. Data are expressed as the mean ± SEM of three independent experiments. ^#P < 0.01, as compared with the cells stimulated with LTA alone. c Scheme of the LTA-mediated signaling pathways linked to MMP-9 expression and cell migration in brain astrocytes. Action of LTA to its receptors (TLR2) results in activation of PKCα/Nox2/ROS cascade. MMP-9 transcription is ATF-2/AP-1-dependently regulated by a ROS-dependent pathway. Moreover, p300 plays the role of an assistant mediator in these responses. This signaling pathway might contribute to sustained expression of MMP-9, which is required for cell migration in RBA-1 cells.