Regulation of the innate immune response by fibronectin: synergism between the III-1 and EDA domains.

Fibronectin is a critical component of the extracellular matrix and alterations to its structure will influence cellular behavior. Matrix fibronectin is subjected to both mechanical and biochemical regulation. The Type III domains of fibronectin can be unfolded in response to increased cellular contractility, included or excluded from the molecule by alternative splicing mechanisms, or released from the matrix by proteolysis. Using Inflammatory Cytokine microarrays we found that the alternatively spliced fibronectin Type III domain, FnEDA, and the partially unfolded III-1 domain, FnIII-1c, induced the expression of a multitude of pro-inflammatory cytokines in human dermal fibroblasts, most notably CXCL1-3, IL-8 and TNF-α. FnIII-1c, a peptide representing an unfolded intermediate structure of the first Type III domain has been shown to initiate the toll-like receptor-4 (TLR4)-NFκB-dependent release of cytokines from human dermal fibroblasts (You, et al., J. Biol. Chem., 2010). Here we demonstrate that FnIII-1c and the alternatively spliced FnEDA domain induce a TLR4 dependent activation of p38 MAP kinase and its downstream effector, MAPKAP Kinase-2 (MK-2), to regulate cytokine expression in fibroblasts. RT-qPCR analysis indicated that the p38-MK-2 pathway regulates IL-8 mRNA stability. Interestingly, addition of FnIII-1c and FnEDA synergistically enhanced TLR4-dependent IL-8 release. These data indicate that Fn contains two Type III domains which can activate TLR signaling to induce an inflammatory response in fibroblasts. Furthermore, our data identifies the NF-κB and p38/MK2 signaling pathways as transducers of signals initiated in response to structural changes in fibronectin

IL-8 expression induced by FnIII domains is NF-κB and TLR4-dependent.

<p>Fibroblasts were serum-starved overnight before treatment with 20 µM FnIII-1c, FnEDA, FnIII-10n, FnIII-13 or PBS in 0.1% BSA-DMEM for the indicated amount of time (A) or for 4 h at the indicated dose (B). Fibroblasts or THP-1 monocytes were incubated with increasing doses of LPS for 4 h (C). Tenascin C (100 µg/ml), LPS (1 µg/ml) or FnEDA (200 µg/ml) were incubated with either dermal fibroblasts or THP-1 monocytes for 4 h (D). Cells were pretreated with the indicated amounts of inhibitor or monoclonal antibody for 1 h prior to the addition of FnIII-1c (E) or FnEDA (F) in 0.1% BSA-DMEM for 4 h. In all experiments, conditioned media was collected and IL-8 protein amounts determined by ELISA. To determine significance, statistical analysis was performed using Student's t test. *p<0.05. Error bars indicate mean ± S.D. of three separate experiments performed in triplicate.</p

Activation of MK-2 by FnIII-1c regulates IL-8 mRNA stability.

<p>Monolayers of dermal fibroblasts were serum-starved overnight and incubated with 10 µM III-1c for 2 h prior to the addition of 10 µM actinomyosin D with or without 20 µM MK-2 inhibitor. After incubation for the indicated times, IL-8 mRNA levels were assessed by RT-PCR. The mRNA level was analyzed relative to the blank control and balanced with the housekeeping gene, β-actin using ΔΔCt (A). The percent (%) change in mRNA expression between 0–30 min and 30–60 min was calculated and analyzed using a two way ANOVA followed by t-test with Bonferroni correction (p<0.05) (B). Error bars indicate mean ± SD of 12 samples. Data are combined from 6 experiments performed in duplicate.</p

Fibronectin Type III domains: Selective induction of cytokines in fibroblasts.

<p>Monolayers of human dermal fibroblasts were serum-starved before treatment with 20 µM FnIII-1c (A), FnEDA (C), FnIII-10n (E), FnIII-13 (F) or 100 µg/ml Tenascin C (G). RNA was extracted and gene expression profiling was performed using a Human Autoimmune and Inflammatory Cytokine PCR array. Dashed lines indicate a 1000 fold change in gene expression (A and C) and a 10 fold increase in gene expression (E, F, G). The 5 genes whose expression was increased ≥1000 fold are shown in tables (B,D).</p

The p38 MAPK/MK-2 pathway regulates FnIII-1c induced IL-8 expression.

<p>Fibroblasts were treated with the indicated concentration of inhibitors for p38 (A, B) or MK-2 (C, D) for 1 h prior to stimulation with 20 µM FnIII-1c. The amount of IL-8 protein present in the medium was measured by ELISA at 4 h (A, C). MK-2 phosphorylation in the presence of increasing amounts of p38 inhibitor (SB203580) was assessed using antibodies to phospho-MK-2 (B). HSP27 phosphorylation in the presence of MK-2 inhibitor was assessed by immunoblotting with antibodies to phospho-HSP27. FAK served as loading control (D). Data are expressed as % control where cells which received 20 µM FnIII-1c without inhibitor (0) in A and C are set at 100%. Error bars indicate mean ± SD of 3 separate experiments.</p

FnIII-1c activates TLR4-dependent p38 MAPK/MK-2 signaling in dermal fibroblasts.

<p>Fibroblasts were serum-starved overnight and treated with the indicated amounts of Fn Type III domains, FnIII-1c, FnIII-10n or FnIII-13 for 1 h. Cell layers were lysed and proteins were electrophoresed and immunoblotted with antibodies to phospho-p38 (A, C) and phospho-MK-2 (B, D). Cells were pretreated with increasing concentrations of antibodies to human TLR4 or 20 µg/ml control antibody, goat IgG (GIgG) for 30 min prior to the addition of 20 µM FnIII-1c for 1 h. Cells were lysed and immunoblotted for phospho-p38 and phospho-MK-2 (E). Immunoblotting for total p38, MK-2 or FAK served as loading controls.</p

FnIII-1c activates NF-κB in parallel with the p38 MAPK signaling pathway.

<p>Monolayers of human fibroblasts were serum-starved overnight and treated with the indicated concentration of p38 inhibitor (SB203580), MK-2 inhibitor (MK-2 inhibitor III), or NFκB inhibitors (PS-1145, Bay11-7082) for 1 h. Cells were then stimulated with 20 µM FnIII-1c for 1vh. Cells were lysed and the nuclear fraction isolated and analyzed by Western blot for the presence of NF-κB protein p65/rel A (A). Blots were quantified by densitometry and normalized to total lamin A/C. Values are mean ± S.D. of 3 separate experiments (B). Cytoplasmic fractions were analyzed by Western blot for p-p38 (C). Membranes were stripped and reprobed with an antibody to nuclear Lamin A/C or FAK, which served as loading control. Proposed signaling pathway (D).</p

FnEDA and FnIII-1c domains work synergistically to regulate IL-8 expression.

<p>Monolayers of human dermal fibroblasts were serum-starved overnight and then incubated with FnIII-1c and FnEDA alone or in combination. After 4 h (A, B, C) or 24 h (D), the amount of IL-8 protein in the conditioned medium was determined by ELISA. Dashed lines and arrows in (C) indicate the approximate Km of FnIII-1c on IL-8 production. Specific amounts of each module are provided in the Figure. Error bars indicate mean ± S.D. of three experiments performed in triplicate.</p