Keratinocyte integrin α3β1 induces expression of the macrophage stimulating factor, CSF-1, through a YAP/TEAD-dependent mechanism

Abstract

The development of wound therapy targeting integrins is hampered by inadequate understanding of integrin function in cutaneous wound healing and the wound microenvironment. Following cutaneous injury, keratinocytes migrate to restore the skin barrier, and macrophages aid in debris clearance. Thus, both keratinocytes and macrophages are critical to the coordination of tissue repair. Keratinocyte integrins have been shown to participate in this coordinated effort by regulating secreted factors, some of which crosstalk to distinct cells in the wound microenvironment. Epidermal integrin α3β1 is a receptor for laminin-332 in the cutaneous basement membrane. Here we show that wounds deficient in epidermal α3β1 express less epidermal-derived macrophage colony-stimulating factor 1 (CSF-1), the primary macrophage-stimulating growth factor. α3β1-deficient wounds also have fewer wound-proximal macrophages, suggesting that keratinocyte α3β1 may stimulate wound macrophages through the regulation of CSF-1. Indeed, using a set of immortalized keratinocytes, we demonstrate that keratinocyte-derived CSF-1 supports macrophage growth, and that α3β1 regulates Csf1 expression through Src-dependent stimulation of Yes-associated protein (YAP)-Transcriptional enhanced associate domain (TEAD)-mediated transcription. Consistently, α3β1-deficient wounds in vivo display a substantially reduced number of keratinocytes with YAP-positive nuclei. Overall, our current findings identify a novel role for epidermal integrin α3β1 in regulating the cutaneous wound microenvironment by mediating paracrine crosstalk from keratinocytes to wound macrophages, implicating α3β1 as a potential target of wound therapy.

Keywords: Csf-1; Integrin α3β1; Keratinocyte; Macrophage; Wound healing; YAP/TAZ.

Figure 1.

α3eKO wounds express less epidermal CSF-1 and have fewer wound-proximal macrophages. Cryosections of 3-day wounds were prepared from control or α3eKO mice as in Fig. S1 and stained by IF with anti-α3 (green, upper and lower panels) and DAPI (blue, lower panels only); arrowheads indicate edge of wound epidermis; scale bars, 25 μm (upper panel), 100 μm (lower panel). (b) IF with anti-CSF-1 (red) and DAPI (blue); dashed line, outlines wound epidermis; scale bar, 500 μm. Graph shows relative CSF-1 mean fluorescence intensity. (c) ISH to detect epidermal-derived Csf1 mRNA was performed on cryosections; Csf1 mRNA (purple) and DAPI (blue); images were taken fully within wound epidermis; scale bar, 25 μm. Graph shows quantification of relative mRNA puncta. (d) IF with anti-F4/80 (red), anti-K14 (green), and DAPI (blue); box indicates area of inset that is magnified on right with (lower) and without (upper) DAPI; arrows, wound-proximal macrophages; scale bar, 50 μm. Graph shows percentage of cells in wound bed that are F4/80-positive. (b–d) Data are mean +/− s.e.m.; n ≥ 5 wounds per genotype; * p<0.05; *** p<0.001. (a,b,d) e, epidermis; d, dermis; s, scab; wb, wound bed.

Figure 2.

Keratinocyte integrin α3β1 promotes CSF-1 expression and supports macrophage growth. (a) Immunoblot of whole cell lysates to validate α3 expression levels in MK cells that lack (α3−/−) or express (α3+/+, α3res) α3β1; ERK, loading control. (b) qPCR quantification of relative Csf1 mRNA levels in MK cells. (c) Representative immunoblot of secreted CSF-1 in CM from MK cells. (d) Quantification of secreted CSF-1 shown in panel c. (e) Macrophage density across 6 consecutive days, represented as fold-change from day 1, when grown in medium conditioned by MK cells; statistical analysis performed on day 6 data (b, d, e). Data are mean +/− s.e.m.; n ≥ 3; * p<0.05; ** p<0.01.

Figure 3.

Macrophage growth is reduced in CM from CSF-1-knockdown keratinocytes. MKα3+/+ cells were treated with one of two distinct siRNAs (#1 or #2) that target Csf1 gene transcripts or a non-targeting siRNA as control. (a) qPCR quantification of Csf1 mRNA levels. (b) Quantification of relative secreted CSF-1, determined by immunoblot of CM from MK cells. (c) Macrophage density across 6 consecutive days, represented as fold-change from day 1, when grown in medium conditioned by MKα3+/+ cells treated with control siRNA (circles), or with CSF-1-targeting siRNA #1 (triangles) or #2 (squares). Statistical analysis was performed on day 6 data. (a–c). Data are mean +/− s.e.m.; n = 3; * p<0.05; ** p<0.01.

Figure 4.

Inhibition of integrin α3β1-dependent YAP/TAZ-TEAD activity reduces Csf1-expression in MK cells. TEAD-dependent transcriptional activity was measured using TEAD-reporter assays on (a) MKα3+/+ versus MKα3−/− cells, or (b) MKα3+/+ cells treated with the TEAD-inhibitor, MGH-CP1, or control (DMSO). For each n, the average normalized luciferase levels (Firefly luciferase/Renilla Luciferase) from duplicate wells is plotted. (c) MKα3+/+ cells were treated with 10uM MGH-CP1 or DMSO (dotted line); gene expression of Csf1, Ctgf, Cyr61, and Bmp1 was determined by qPCR. (a–c) Data are mean +/− s.e.m.; n = 4; * p<0.05; ** p<0.01; *** p<0.001; ns, not significant.

Figure 5.

YAP activation in MKα3−/− cells rescues the expression of Csf1 and other YAP/TAZ target genes in a TEAD-dependent manner. (a–e) MKα3−/− cells were transfected with empty expression vector (control), or vector that expresses YAP S127A, TAZ S89A, or YAP-S127A/S94A (see Materials and Methods for details). (a) TEAD-dependent transcriptional activity was measured using TEAD-reporter assays. For each n, the average normalized luciferase levels (Firefly luciferase/Renilla Luciferase) from duplicate wells is plotted. (b–e) qPCR was performed to assay gene expression of (b) Ctgf, (c) Cyr61, (d) Csf1, or (e) Bmp1. Data are mean +/− s.e.m.; n = 4; ** p<0.01; *** p<0.001; **** p<0.0001; ns, not significant.

Figure 6.

Inhibition of integrin α3β1-dependent Src activity in MK cells reduces the expression of Csf1 and other YAP/TAZ target genes. (a,b) Whole cell lysates were prepared from MKα3+/+ cells treated with (a) PP2 or (b) dasatinib, or equal volume of DMSO as control, then immunoblotted for active Src (pSrc Y416), total Src (tSrc), phospho-FAK (pFAK Y925) or GAPDH. Quantitation relative to DMSO and normalized to GAPDH is reported below blots as an average of three experiments. (c-f) MKα3+/+ cells were treated with PP2 (c,e) or dasatinib (d,f) versus DMSO as control. (c,d) TEAD-dependent transcriptional activity was measured using TEAD-reporter assays. For each n, the average normalized luciferase levels (Firefly luciferase/Renilla Luciferase) from duplicate wells is plotted. (e,f) Relative gene expression of Csf1, Ctgf, and Cyr61, was determined by qPCR. Data are mean +/− s.e.m.; n = 3; * p<0.05; *** p<0.001; **** p<0.0001; ns, not significant.

Figure 7.

Integrin α3β1-deficient wound epidermis has a reduced number of keratinocytes with YAP-positive nuclei. Cryosections of 3-day wounds were prepared as in Figure 1. (a) Representative cryosections stained with anti-YAP (red) and DAPI (blue); e, epidermis; wb, wound bed; dashed line, underlines wound epidermis; scale bar, 50 μm. (b) Graph shows percentage of wound keratinocytes with YAP-positive nuclei. Data are mean +/− s.e.m.; n ≥ 7 wounds per genotype; ** p<0.01. (c) Model: α3β1 on wound keratinocytes activates FAK-Src signaling that promotes YAP-TEAD-dependent transcription of Csf1. CSF-1 protein is secreted from the wound epidermis to support macrophages of the wound bed.