Integrin α3β1 in hair bulge stem cells modulates CCN2 expression and promotes skin tumorigenesis

(A) Overview of the K14 Itga3 KO and WT mouse models. (B) Whole mounts of tail epidermis show the presence of α3β1-depleted K15-positive keratinocytes in upper parts of hair follicles and IFE of K14 Itga3 KO mice (white arrow heads). Remaining α3β1-positive keratinocytes in K14 Itga3 KO mice are preferentially localized to HBs (scale bar: 100 μm). (C) Staining for integrin α3 shows HB localization of α3β1-positive keratinocytes in the back skin of 7-wk-old K14 Itga3 KO mice. α3β1 is found in all basal keratinocytes of K14 Itga3 WT mice of similar age (scale bar: 50 μm). (D) FACS analysis of keratinocytes isolated from back skin epidermis. The chart shows the percentages of α3-positive HB cells (CD34-positive) in the total α3-positive population. Each dot represents a mouse. Gating strategy can be found in Fig S2A (mean ± SD, unpaired t test, *P < 0.05). (E) GAPDH-normalized relative mRNA expression of K15 is increased in the epidermis of back and tail skin of K14 Itga3 KO compared with WT mice. Each dot represents a mouse and is an average of technical duplicate or triplicate (mean ± SD, unpaired t test, *P < 0.05, **P < 0.005).

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(A, B) H&E staining (left) and quantification (right) of wound healing. (A, B) Wound closure is comparable between K19 Itga3 KO and WT mice, 3 (A) and 5 d (B) after wounding. (A) Each dot represents the average length of the neo-epidermis (black arrows) per wound (mean ± SD, unpaired t test). Wounds of six K19 Itga3 WT and five K19 Itga3 KO mice were analyzed (scale bar: 300 μm). (B) Bars represent the percentage of closed wounds per mouse (mean ± SD). Wounds of four K19 Itga3 WT and four K19 Itga3 KO mice were analyzed (scale bar: 1 mm). (C, D) Immunohistochemistry (IHC) staining for GFP (left) and quantification (right) of GFP-positive area per neo-epidermis of K19 Itga3 KO and WT mice. (C) HB-originating GFP-positive keratinocytes comparably contribute to neo-epidermis in K19 Itga3 KO and WT mice 3 d after wounding (scale bar: 300 μm). Each dot represents the percentage of GFP-positive area per wound (mean ± SD, unpaired t test). Wounds of six K19 Itga3 WT and five K19 Itga3 KO mice were analyzed. (D) 5 d after the wounding, the contribution of the α3β1-deficient HB SCs to the newly formed epidermis is more extensive than that of the α3β1-proficient HB SCs. Each dot represents the percentage of GFP-positive area per wound (mean ± SD, unpaired t test, *P < 0.05). Wounds of five K19 Itga3 WT and four K19 Itga3 KO mice were analyzed.

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(A) The number of tumors (left) and tumor burden (right) is decreased in K19 Itga3 KO compared with WT mice submitted to the DMBA/TPA carcinogenesis protocol (mean ± SEM, unpaired t test, *P < 0.05, **P < 0.005, ***P < 0.0005). (B) Representative macro images of K19 Itga3 KO and WT mice at the end of the treatment. (C) Histology of benign papillomas and keratoacanthomas, representing the majority of tumors isolated from K19 Itga3 KO and WT mice.

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(A, B) With rare exceptions, Cre-induced GFP-positive cells represent less than 1% of total tumor mass. (A, B) Representative IHC images stained for GFP and (B) quantification of GFP-positive area in cross sections of tumors, isolated from nine K19 Itga3 KO and eight WT mice. The vast majority of tumors is GFP negative (scale bar: 1 mm). (C) Integrin α3β1 is strongly expressed in all tumors analyzed (scale bar: 1 mm). (D, E) Analysis of GFP-positive area over 10 cross sections, cut every 200 μm of randomly selected tumors from four K19 Itga3 KO and four WT mice. (D) The contribution of GFP-positive HB-originating keratinocytes to tumors, isolated from K19 Itga3 KO is significantly reduced compared with WT mice (mean ± SEM, unpaired t test, *P < 0.05). (E) Most tumors are GFP negative in both, K19 Itga3 KO and WT mice. GFP was detected in 9.6% of K19 Itga3 KO and in 32.3% of K19 Itga3 WT tumors and did not exceed 1% of total tumor mass.

(A) GFP-positive Cre-induced HB SCs localize to growing hair follicles (HFs) and, in some cases, to isthmus, infundibulum, and IFE (black arrows) after short-term DMBA/TPA treatment in K19 Itga3 KO and WT mice. Left: IHC staining for GFP (scale bar: 100 μm). Right: quantification of the number of HFs, where GFP-positive cells were observed in the upper parts of HFs and in adjacent IFE. Each dot represents a mouse (mean ± SD, unpaired t test). (B) Heat map of row-scaled significantly differentially expressed protein-coding genes of GFP-positive keratinocytes, isolated from three K19 Itga3 KO and three K10 Itga3 WT mice after short-term DMBA/TPA treatment. Protein-coding genes have an adjusted P < 0.05 and an average normalized expression across all samples >4 (as calculated with Voom) and a logFC > 0.6 between K19 Itga3 WT an KO mice. (C) IF staining (left) and quantification of mean intensity of the signal (right) for CCN2 in GFP-positive HFs after short-term DMBA/TPA treatment. Each dot represents a GFP-positive HF. HFs of five K19 Itga3 KO and six K19 Itga3 WT mice were quantified (mean ± SD, unpaired t test, P < 0.0001).

(A) GAPDH-normalized relative mRNA expression of CCN2 is significantly decreased in non-stimulated as well as IL-6 and TPA-treated α3β1-depleted keratinocytes. The average of up to four independent measurements of technical duplicates of four RNA samples per group (dots) is presented (mean ± SD, Fisher’s LSD test, *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001). (B) IF (left) and quantification of the mean intensity (right) of CCN2 in non-stimulated, IL-6, and TPA-treated MSCC Itga3 KO and WT keratinocytes. Expression of CCN2 is α3β1 dependent and increases upon IL-6 and TPA treatment (scale bar: 50 μm). 90 cells imaged over three independent experiments were quantified (mean ± SD, Fisher’s LSD test, P < 0.0001). (C) Representative WB confirming α3β1-dependent and IL-6– and TPA-mediated CCN2 expression. Quantification can be found in Fig S5A.

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(A) WB of CCN2 and integrin α3-expression of selected CCN2 KO and control clones. (B) Representative image (top) and quantification (bottom) of colony-formation assay of MSCC Itga3 WT and Itga3 KO cells and MSCC CCN2 KO G1, KO G2, control G1, and control G2 clones. Deletion of α3β1 results in a strong reduction of colony formation and colony size. Moderate reduction of colony formation can be seen upon CCN2 deletion. Quantification of colony size can be found in Fig S6A. Average values of technical triplicates of five independent experiments are presented (mean ± SD, Fisher’s LSD test, *P < 0.05, **P < 0.005, ****P < 0.0001). (C) Representative image (top) and quantification (bottom) of colony-formation assay of MSCC CCN2 KO G1 and KO G2 clones, grown in control conditions or in the presence of 45 ng/ml CCN2, as well as CCN2 WT control G1 and control G2 clones. Treatment with exogenous CCN2 significantly increases colony formation of CCN2 KO clones. Quantification of colony size can be found in Fig S6B. Average values of technical triplicates of four independent experiments are presented (mean ± SD, Fisher’s LSD test, *P < 0.05, **P < 0.005). (D) No differences in the number of colonies can be observed upon CCN2 treatment of Itga3 KO–transformed keratinocytes. Quantification of colony size can be found in Fig S6B. Average values of technical triplicates of four independent experiments are presented (mean ± SD, Fisher’s LSD test, ****P < 0.0001). (E) Quantification (left) and IF as maximum intensity projection (right) of CCN2 expression of MSCC Itga3 WT and KO spheroids, grown in 3D Matrigel matrix for 1 or 7 d (scale bar: 20 μm). The expression of CCN2 is α3β1 dependent, which is particularly prominent at the beginning of spheroid growth. The percentage of CCN2-positive area was quantified from 17 MSCC Itga3 KO and 30 MSCC Itga3 WT spheroids 1 d after seeding and from 15 MSCC Itga3 KO and 27 MSSC WT spheroids 7 d after seeding (mean ± SEM, unpaired t test, *P < 0.05). (F) Spheroid growth in 3D Matrigel is α3β1 dependent and moderately reduced upon CCN2 deletion. Top: bright-filed images of representative spheroids (scale bar: 50 μm). Bottom: size quantification of 60–80 spheroids measured over three to four independent experiments (mean ± SD, Fisher’s LSD test, ****P < 0.0001). (G) 3D growth of CCN2 KO MSCC spheroids shows small but significant increase when cells are seeded with 45 ng/ml of CCN2. Left: bright-filed images of representative spheroids (scale bar: 50 μm). Right: size quantification of 85–90 spheroids measured over three independent experiments (mean ± SD, Fisher’s LSD test, **P < 0.005, ***P < 0.0005 ****P < 0.0001). (H) Seeding Itga3 KO MSCCs with 45 or 180 ng/ml of CCN2 does not impact the 3D growth pf spheroids. Left: bright-filed images of representative spheroids (scale bar: 50 μm). Right: size quantification of 70 spheroids measured over two independent experiments (mean ± SD, one-way ANOVA, P = 0.9491).

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