21 research outputs found
Reciprocal Regulation of Reactive Oxygen Species and Phospho-CREB Regulates Voltage Gated Calcium Channel Expression during <i>Mycobacterium tuberculosis</i> Infection
<div><p>Our previous work has demonstrated the roles played by L-type Voltage Gated Calcium Channels (VGCC) in regulating <i>Mycobacterium tuberculosis</i> (<i>M. tb</i>) survival and pathogenesis. Here we decipher mechanisms and pathways engaged by the pathogen to regulate VGCC expression in macrophages. We show that <i>M. tb</i> and its antigen Rv3416 use phospho-CREB (pCREB), Reactive Oxygen Species (ROS), Protein Kinase C (PKC) and Mitogen Activated Protein Kinase (MAPK) to modulate VGCC expression in macrophages. siRNA mediated knockdown of MyD88, IRAK1, IRAK2 or TRAF6 significantly inhibited antigen mediated VGCC expression. Inhibiting Protein Kinase C (PKC) or MEK-ERK1/2 further increased VGCC expression. Interestingly, inhibiting intracellular calcium release upregulated antigen mediated VGCC expression, while inhibiting extracellular calcium influx had no significant effect. siRNA mediated knockdown of transcription factors c-Jun, SOX5 and CREB significantly inhibited Rv3416 mediated VGCC expression. A dynamic reciprocal cross-regulation between ROS and pCREB was observed that in turn governed VGCC expression with ROS playing a limiting role in the process. Further dissection of the mechanisms such as the interplay between ROS and pCREB would improve our understanding of the regulation of VGCC expression during <i>M. tb</i> infection.</p></div
Rv3416 upregulates VGCC in a TLR dependent pathway.
<p>PMA stimulated THP1 cells were transfected with siRNA against MyD88, TRAF6, IRAK1, IRAK2 or IRAKM for 36h, followed by stimulation with 30 µg/ml of Rv3416 for 72h. L-type VGCC levels were monitored using flow cytometry. Bold lines represent cells transfected with control siRNA followed by stimulations with Rv3416, while dotted lines represent cells transfected with specific siRNA to indicated molecules followed by stimulations with Rv3416. Thin lines represent unstimulated cells transfected with control siRNA. One of three independent experiments is shown. Bar chart shows the Fold increase in Mean Fluorescence Intensity (ΔMFI) in different groups over and above unstimulated control (represented as MOCK). Bars represent mean ± S.D. of three independent experiments.</p
Genes in the calcium/calmodulin and cysteine protease pathways positively regulate ROS levels.
<p>For Panel A, PMA stimulated THP1 cells were transfected with siRNA against indicated genes and ROS levels were monitored by FACS 2h post stimulation with Rv3416. Bold lines represent cells transfected with siRNA against indicated genes while thin lines represent unstimulated cells transfected with control siRNA. Panel B shows pCREB levels in nuclear extracts of PMA stimulated THP1 cells transfected with siRNA against indicated genes followed by stimulation with Rv3416 for indicated times. One of two independent experiments is shown.</p
Rv3416 induces the upregulation of L-type VGCC on macrophages.
<p>THP1 cells were stimulated overnight with 50/ml PMA followed by Rv3416 stimulation for 72h. L-type VGCC expression was monitored by flow cytometry. Bold lines represent stimulations with Rv3416 while thin lines represent unstimulated controls. Panels A-D represent stimulation with 15 µg/ml, 30 µg/ml, 45 µg/ml and 60 µg/ml of Rv3416, respectively. Data from one of four independent experiments are shown.</p
PKC, ERK/MAPK, CAMKII and intracellular calcium release negatively regulate Rv3416 mediated VGCC expression.
<p>PMA stimulated THP1 cells were incubated with inhibitors to indicated molecules (<i>see Experimental Procedures</i>) for 1h prior to stimulation with Rv3416. L-type VGCC levels were monitored by FACS 72h post stimulation. Bold lines represent cells stimulated with 30 µg/ml of Rv3416, while dotted lines represent cells incubated with inhibitors to indicated molecules followed by stimulations with Rv3416. Thin lines represent unstimulated cells. One of four independent experiments is shown.</p
Specific genes in the calcium-calmodulin and cysteine protease pathways differentially regulate Rv3416 mediated VGCC expression.
<p>PMA stimulated THP1 cells were transfected with siRNA against CAMKIIA, Cathepsin H, Prkaa2, SENP8, SNRK or USP25 for 36h, followed by stimulation with 30 µg/ml of Rv3416 for 72h. VGCC levels were monitored using flow cytometry. Bold lines represent cells transfected with control siRNA followed by stimulations with Rv3416, while dotted lines represent cells transfected with specific siRNA to indicated molecules followed by stimulations with Rv3416. Thin lines represent unstimulated cells transfected with control siRNA. One of three independent experiments is shown.</p
Inhibition of PKC or MAK-ERK enhances ROS generation in Rv3416 stimulated cells.
<p>For Panel A, PMA stimulated THP1 cells were incubated with an inhibitor to PKC (subpanel a) or MAPK-ERK (subpanel b) for 1h prior to stimulation with 30 µg/ml of Rv3416 for 2h and ROS levels were monitored by flow cytometry. Thin line represents unstimulated control while bold lines represent Rv3416 stimulated cells. For Panel B and C, PMA stimulated THP1 cells were incubated with inhibitor to PKC (Calphostin C, Panel B) or MAPK-ERK (U0126, Panel C) and stimulated with Rv3416 for indicated times. Nuclear extracts were probed for pCREB levels. Numbers below the blots indicate relative intensities of the bands. Data from one of three experiments is shown.</p
CREB, c-Jun and SOX5 positively regulate Rv3416 mediated VGCC expression on macrophages.
<p>For Panel A, PMA stimulated THP1 cells were stimulated with 30 µg/ml of Rv3416 for indicated times and the levels of indicated transcription factors were monitored in nuclear extracts by western blots. Numbers below the blots indicate relative intensities of the bands. Arrow indicates the specific band based on molecular weight markers. Data from one of two experiments are shown. For Panel B, PMA stimulated THP1 cells were transfected with siRNA against c-Jun (sicJun), CREB (siCREB), NF-KBp65 (siNF-κB) and SOX5 (siSOX5) for 36h, followed by stimulation with 30 µg/ml of Rv3416 for 72h. VGCC levels were monitored using flow cytometry. Bold lines represent cells transfected with control siRNA followed by stimulations with Rv3416, while dotted lines represent cells transfected with specific siRNA to indicated transcription factors followed by stimulations with Rv3416. Thin lines represent unstimulated cells transfected with control siRNA. One of two independent experiments is shown.</p
Rv3416 synergizes with Nef sufficient HIV but not Nef deficient HIV to inhibit apoptosis in macrophages.
<p>PMA stimulated THP1 cells were stimulated with 1 μg/ml Pam3CSK4 (Pam) and infected with 0.5 MOI of wild type HIV or Nef deficient HIV (HIVδNef) or Vpu deficient HIV (HIVδVpu) in the presence or absence of 20 μg/ml Rv3416 for 48h. Cells were stained with Annexin V-APC. Thin lines represent stimulation with Pam3CSK4 alone and the thick lines represent stimulation as indicated. Data from one of three independent experiments are shown. Bar graphs show relative MFIs of the histograms. Data from one of three experiments are shown. P<0.03 for Pam vs Pam+Rv3416. P<0.04 for Pam vs Pam+HIV, P<0.03for Pam vs Pam+Rv3416+HIV.</p
Reciprocal regulation of oxidative burst and calcium routing regulate apoptosis in macrophages by Rv3416 and Nef.
<p>For Panel A and B, PMA stimulated THP1 cells were treated with 50 μM H<sub>2</sub>O<sub>2</sub> for 1h and stimulated with 1 μg/ml Pam3CSK4 along with 20 μg/ml Rv3416 and 15 μg/ml Nef for 24h. For Panel A, cells were stained with Annexin V-APC. Thin lines represent cells stimulated in the absence of H<sub>2</sub>O<sub>2</sub> while the thick lines represent cells stimulated in the presence of H<sub>2</sub>O<sub>2</sub>. Data from one of three independent experiments are shown. Bar graphs adjacent to histogram in Panel A show relative MFIs of the histogram. For Panel B, PMA stimulated THP1 cells were stimulated with 1 μg/ml Pam3CSK4 along with 20 μg/ml Rv3416 or 15 μg/ml Nef or both for 24h and cytoplasmic extracts were probed for indicated molecules and analyzed by western blots. Numbers below the blots indicate the relative intensities of the bands. Data from one of three experiments are shown. For Panel C (PMA stimulated THP1 macrophages) and Panel E (Blood macrophages) were stimulated with 1 μg/ml Pam3CSK4 along with 20 μg/ml Rv3416 (dotted lines) or 15 μg/ml Nef (thin lines) or both (thick lines) for 2h for measuring oxidative burst. Thirty minutes prior to the incubation period, cells were loaded with 10μM DCFH-DA. At the end of the incubation period, cells were thoroughly washed with the culture medium and immediately analyzed for ROS levels by flow cytometry. Shaded histograms in Panel C and E represent cells stimulated with 1 μg/ml Pam3CSK4 only. Bar graphs adjacent to histograms in Panel C and Panel D show relative MFIs of the corresponding histograms. For Panel D (THP-1 macrophages) and Panel F (Blood macrophages) cells stimulated with 1 μg/ml Pam3CSK4 along with 20 μg/ml Rv3416 and 15 μg/ml Nef with or without EGTA or TMB-8 for 1h and ROS levels were measured. In Panel C P<0.004 for Pam vs Pam+Rv3416+Nef; In Panel D, P<0.005 for Pam+Rv3416+Nef vs Pam+Rv3416+Nef+TMB; P<0.005 for Pam+Rv3416+Nef vs Pam+Rv3416+Nef+EGTA. In Panel E, P<0.03 for Pam vs Pam+Rv3416+Nef; In Panel F, P<0.05 for Pam+Rv3416+Nef vs Pam+Rv3416+Nef+TMB; P<0.04 for Pam+Rv3416+Nef vs Pam+Rv3416+Nef+EGTA.</p