TLR agonists regulate PDGF-B production and cell proliferation through TGF-beta/type I IFN crosstalk

Abstract

Transforming growth factor-beta (TGF-beta) and type I interferon (IFN) autocrine/paracrine loops are recognized as key mediators of signaling cascades that control a variety of cellular functions. Here, we describe a novel mechanism by which Toll-like receptor (TLR) agonists utilize these two autocrine/paracrine loops to differentially regulate the induction of PDGF-B, a growth factor implicated in a number of diseases ranging from tumor metastasis to glomerulonephritis. We demonstrate that CpG-specific induction of PDGF-B requires activation of Smads through TGFbeta1 autocrine/paracrine signaling. In contrast, polyinosinic:polycytidylic acid strongly represses CpG's as well as its own intrinsic ability to induce PDGF-B mRNA through type I IFN-mediated induction of Smad7, a negative regulator of Smad3/4. Furthermore, we have shown that this crosstalk mechanism translates into similar regulation of mesangial cell proliferation. Thus, our results demonstrate the importance of crosstalk between TGF-beta and type I IFNs in determining the specificity of TLR-mediated gene induction.

Figure 1

CpG specifically upregulates PDGF-B mRNA in a MyD88-IRAK4-dependent manner. (A) BMMs were stimulated with CpG (100 nM), polyI:C (1 μg/ml) or lipid A (1 ng/ml) for 4 h. Total RNA was collected and subjected to microarray analysis as described in Materials and methods. Values in graph are average difference change versus media. (B) BMMs were stimulated with CpG (100 nM) or polyI:C (1 μg/ml) for 4 h. RNA was collected and analyzed by Q-PCR. (C) BMMs were stimulated with CpG (100 nM). Whole-cell lysate was collected and analyzed by Western blot with anti-PDGF-BB or anti-actin. (D) BMMs (WT, MyD88−/−, IRAK4−/−) were stimulated with CpG (100 nM) or polyI:C (1 μg/ml) for 4 h. RNA was collected and analyzed by Q-PCR.

Figure 2

Smad4 is recruited to the PDGF-B promoter by CpG but not polyI:C. (A) Diagram of PDGF-B and IFN-β promoters. (B, C) BMMs were stimulated with CpG (100 nM) or polyI:C (1 μg/ml) for 3 h. Cells were fixed with formaldehyde. ChIP with anti-Smad4 or anti-p65 was performed on sonicated samples, washed thoroughly and analyzed by PCR/gel electrophoresis and Q-PCR, normalized to input.

Figure 3

CpG induction of PDGF-B mRNA is regulated by Smads and NF-κB. (A) BMMs were stimulated with polyI:C (1 μg/ml), CpG (100 nM) or TGFβ1 (2.5 ng/ml) for 4 h. RNA was collected and analyzed by Q-PCR. (B) Raw-WT and Raw-IκB-DA cell lines were pretreated with tamoxifen (200 nM) for 2 h and stimulated with CpG (100 nM) or polyI:C (1 μg/ml) for 4 h. RNA was collected and analyzed by Q-PCR.

Figure 4

CpG more potently activates Smad3/4 than polyI:C through TGF-β. (A) BMMs were stimulated with CpG (100 nM), polyI:C (1 μg/ml) or TGFβ1 (2.5 ng/ml). Nuclear extract was collected and analyzed by EMSA with Smad3/4 consensus probe (Santa Cruz). CpG sample was used for 100 × WT/Mut cold competition. Supershift experiments on samples stimulated with CpG (100 nM) were carried out with anti-Flag (M2) or mouse IgG. (^*) Flag-Smad3 supershifted complex; (^**) native complex. (B) RAW 264.7 cells were transfected with 1 μg 3TP-Luc or κB-Luc and stimulated with CpG (500 nM), TGFβ1 (5 ng/ml) or polyI:C (5 μg/ml) for 24 h. Luciferase activity was measured and normalized to β-galactosidase activity. Graph shown is the average of three independent experiments. (C) BMMs were stimulated with CpG and anti-TGF-β neutralizing antibody or control IgG (2 μg/ml) for 4 h. RNA was collected and analyzed by Q-PCR. (D) BMMs were serum starved for 6 h and then stimulated with CpG (500 nM), LPS (10 ng/ml) or polyI:C (1 μg/ml) in serum-free media for 24 h. The supernatant was analyzed by ELISA for TGFβ1. (E) Raw-WT or Raw-IκB-DA cells were serum starved overnight and stimulated with CpG (100 nM) in serum-free media for 24 h. The supernatant was analyzed by ELISA for TGFβ1.

Figure 5

PolyI:C inhibits induction of PDGF-B mRNA through activation of the type I IFN pathway. (A) BMMs were stimulated with CpG (100 nM) and increasing amounts of polyI:C (0, 1 and 10 μg/ml). RNA was collected and analyzed by Q-PCR. (B) BMMs were stimulated with CpG and polyI:C (1 μg/ml) or recombinant IFN-β (200 U/ml). RNA was collected and analyzed by Q-PCR. (C) IFNAR+/+ and IFNAR−/− BMMs were stimulated with CpG (100 nM), polyI:C (1 μg/ml) or both for 4 h. RNA was collected and analyzed by Q-PCR. (D) Repeated (C) with Stat1+/+ and Stat1−/− BMMs. (E) BMMs were stimulated with TGFβ1 (2.5 ng/ml) and polyI:C (1 μg/ml) or recombinant IFN-β (200 U/ml). RNA was collected and analyzed by Q-PCR.

Figure 6

PolyI:C repression of CpG-mediated Smad3/4 activation and PDGF-B mRNA induction is mediated through Smad7. (A) BMMs were stimulated with CpG (100 nM), polyI:C (1 μg/ml) or both for 3 h. Nuclear extract was collected and analyzed by EMSA with Smad3/4. Media sample was used for 100 × WT/Mut cold competition. (B) IFNAR+/+ and IFNAR−/− BMMs were treated for 30 min with CM collected from IFNAR+/+ BMMs that were stimulated with CpG (100 nM) or polyI:C (1 μg/ml) for 2 h. STAT1+/+ and STAT1−/− BMMs were stimulated with STAT1+/+ BMM CM for 4 h. RNA was collected and analyzed by Q-PCR. (C) Raw-MT and Raw-Flag-Smad7 cell lines were stimulated with CpG (100 nM) and TGFβ1 (2.5 ng/ml) for 4 h. RNA was collected and analyzed by Q-PCR. Western blot with anti-Flag (M2) or anti-actin was performed to confirm expression of Flag-Smad7. (D) Raw-MT and Raw-Flag-Smad7-DN cell lines were stimulated with CpG (100 nM) with or without polyI:C (1 μg/ml). RNA and protein expressions were carried out as in (C). RAW 264.7 cell lines were transfected with Smad7 or nonspecific oligos (100 nmol). At 36 h after transfection, RNA and protein levels were analyzed.

Figure 7

Maximal CpG induction of mesangial cell proliferation involves PDGF-BB. (A) Mesangial cells were serum starved for 3 days and then stimulated for 48 h with CM from BMMs stimulated overnight with CpG (100 nM), polyI:C (1 μg/ml) or both. [³H]thymidine was added 6 h prior to collection. Cells were harvested and analyzed by scintillation counter. Graphs are the average of three independent experiments. (B) CM from BMMs stimulated overnight with CpG (100 nM) was thoroughly mixed with anti-PDGF-BB neutralizing antibody or control IgG (0, 1 or 10 μg/ml) and used to stimulate mesangial cells as in (A). (C) Model of CpG and polyI:C regulation of PDGF-B. CpG and polyI:C induce TGF-β production through NF-κB activation. TGFβ1 autocrine/paracrine loop activates Smad3/4, which induces PDGF-B. PolyI:C also induces the IFN-β autocrine/paracrine loop, activating Stat1 and inducing Smad7. Smad7 inhibits polyI:C activation of Smad3/4, prohibiting PDGF-B induction.