A β2-Integrin/MRTF-A/SRF Pathway Regulates Dendritic Cell Gene Expression, Adhesion, and Traction Force Generation

Abstract

β2-integrins are essential for immune system function because they mediate immune cell adhesion and signaling. Consequently, a loss of β₂-integrin expression or function causes the immunodeficiency disorders, Leukocyte Adhesion Deficiency (LAD) type I and III. LAD-III is caused by mutations in an important integrin regulator, kindlin-3, but exactly how kindlin-3 regulates leukocyte adhesion has remained incompletely understood. Here we demonstrate that mutation of the kindlin-3 binding site in the β2-integrin (TTT/AAA-β2-integrin knock-in mouse/KI) abolishes activation of the actin-regulated myocardin related transcription factor A/serum response factor (MRTF-A/SRF) signaling pathway in dendritic cells and MRTF-A/SRF-dependent gene expression. We show that Ras homolog gene family, member A (RhoA) activation and filamentous-actin (F-actin) polymerization is abolished in murine TTT/AAA-β2-integrin KI dendritic cells, which leads to a failure of MRTF-A to localize to the cell nucleus to coactivate genes together with SRF. In addition, we show that dendritic cell gene expression, adhesion and integrin-mediated traction forces on ligand coated surfaces is dependent on the MRTF-A/SRF signaling pathway. The participation of β2-integrin and kindlin-3-mediated cell adhesion in the regulation of the ubiquitous MRTF-A/SRF signaling pathway in immune cells may help explain the role of β2-integrin and kindlin-3 in integrin-mediated gene regulation and immune system function.

Keywords: LAD-III; MKL-1; MRTF-A; SRF; adhesion; dendritic cells; traction force.

Figure 1

Abolished β2-integrin/kindlin-3 link leads to impaired actin dynamics and impaired nuclear MRTF-A shuttling. (A) RhoA activity in WT and KI dendritic cells was measured in serum starved cells (0.3% fetal calf serum (FCS)/RPMI for 1 h) and serum-stimulated cells (15% FCS for 10 min). N = 3, one representative result is shown. (B) F-actin content was assessed via measurement of corrected total cell fluorescence (CTCF) of TRITC-phalloidin stained cells. Baseline level of F-actin was acquired from serum starved cells and F-actin fold change was calculated for 15% FCS-stimulated cells (30 min stimulation). 25–100 cells per condition were measured, N = 3. (C) Immunofluorescence images of serum starved and serum-stimulated WT and KI cells stained with TRITC-phalloidin. *p < 0.05.

Figure 2

Abolished β2-integrin mediated adhesion leads to impaired nuclear MRTF-A shuttling. (A) Total percentages of dendritic cells with nuclear MRTF-A and cytoplasmic MRTF-A are shown. N = 4 and 200 cells per condition were analyzed. MRTF-A staining was performed on non-starved, starved and serum stimulated cells and (B) immunofluorescence images of WT and KI cells are shown after serum stimulation. (C) F-actin content as CTCF of WT and kindlin-3^−/− 25–100 dendritic cells per condition were measured, N = 2. (D) Total percentages of kindlin-3^−/− and WT dendritic cells with nuclear MRTF-A and cytoplasmic MRTF-A are shown. (E) Total percentages of adhesion on iC3b induced nuclear MRTF-A and cytoplasmic MRTF-A are shown. WT and KI dendritic cells were detached, serum starved in suspension, and stimulated with replating on glass coverslips or on iC3b coated coverslips. (F) Total percentages of WT dendritic cells starved on glass compared to KI dendritic cells seeded overnight on fibronectin with nuclear MRTF-A and cytoplasmic MRTF-A. KI dendritic cells were serum starved and stimulated. (A–F) If not otherwise indicated: N = 3 and 200 cells per condition were analyzed. MRTF-A staining was performed on non-starved, starved, and serum stimulated cells.

Figure 3

Inhibition but not deletion of MRTF-A leads to altered CCR7 expression and no change in migration speed. (A) CD40 (B) CD80 (C) CD86 and (D) CRR7 expression in WT NT and MRTF-A inhibited dendritic cells (N = 6); (E) CCR7 expression of MRTF-A ⁻/⁻ and MRTF-A^+/+ (N = 5); surface marker expression was determined by flow cytometry. (F) CCR7 mRNA level of MRTF-A inhibited and NT WT dendritic cells determined by qPCR (N = 4). (G) Scatter plots of tracked WT NT and WT MRTF-A inhibited and (H) MRTF-A⁻/⁻ and MRTF-A^+/+ dendritic cell speeds in a 3D collagen matrix toward CCL19 are shown. Only cells faster than 1 μm/min have been evaluated as migratory and used for the analysis. *p < 0.05.

Figure 4

Gene expression profile of MRTF-A^−/− cells and comparison with integrin KI. (A) Depicted is the overlap of differently regulated genes shared by the MRTF-A⁻/⁻ vs. ^+/+ and the KI vs. WT RNA-Seq data. (B) Ranked list of negative logarithm of adjusted p-values from the reactome pathway enrichment analysis. Pathway was performed using g:Profiler. (C) Node map of pathway enrichment analysis for GO biological process of the GSEA ranked gene list. Analysis is based on RNA-Seq data derived from MRTF-A⁻/⁻ and ^+/+ (N = 4). Nodes highlighted in red are associated with the cytoskeleton. Nodes highlighted with yellow outline contain word “cytoskeleton.” (D) Depiction of cytoskeletal genes that are differently regulated in KI and MRTF-A⁻/⁻.

Figure 5

Inhibition of the MRTF-A/SRF pathway leads to reduced adhesion independent of β2-integrin expression. The effect of MRTF-A inhibition on dendritic cell adhesion to (A) ICAM-1 and (B) to iC3b was compared to adhesion of TTT/AAA-β2-integrin KI cells. Adhesion of resting, non-treated (NT) WT dendritic cells was set to 1 and fold changes for all other conditions was calculated, N = 5. (C) MRTF-A ⁻/⁻ dendritic cell adhesion to ICAM-1 and iC3b was compared to adhesion of MRTF-A^+/+ littermates, N = 4. (D) mRNA level of MRTF-A and MRTF-B in MRTF-A^+/+ mice (N = 4) based on RNA-Seq data. (E) TTT/AAA rescue experiment with constitutively active MRTF-A (MRTF-A^***). Adhesion of non-treated COS-1 cells was set to 1 and fold change for adhesion of COS-1 cells transfected with CD11b/CD18, CD11b/CD18-TTT/AAA (CD18 featuring the TTT/AAA mutation), and CD11b/CD18-TTT/AAA plus MRTF-A^***. (F) Depiction of conditions used in rescue experiment. (G) CD11a, (H) CD11b, (I) CD11c and (J) CD18 expression in WT NT and MRTF-A inhibited dendritic cells (N = 6). (K) CD11a expression in MRTF-A⁻/⁻ and^+/+ dendritic cells (N = 5). (L) CD11a mRNA level of MRTF-A inhibited and NT WT dendritic cells determined by qPCR (N = 4). *p < 0.05, **p < 0.01.

Figure 6

Traction force generation in dendritic cells is regulated by the β2-integrin/MRTF-A/SRF pathway. (A) Heatmaps depicting traction force generated by WT and TTT/AAA KI dendritic cells and (C) non treated WT and MRTF-A inhibited WT dendritic cells. (B) Scatter plots of analyzed WT and KI dendritic cells and (D) non treated WT and MRTF-A inhibited WT dendritic cell traction forces (Pa) are shown. Three experiments were performed, 25 cells were measured each time and a total of 75 cells analyzed. (E) Heatmaps depicting traction force generated by an example MRTF-A⁺/⁺ and ^−/− dendritic cells. (F) Scatter plots of analyzed MRTF-A⁺/⁺ and ^−/− dendritic cells N = 4. *p < 0.05, ***p < 0.005.