RhoA controls retinoid signaling by ROCK dependent regulation of retinol metabolism

Abstract

The ubiquitously expressed small GTPase RhoA is essential for embryonic development and mutated in different cancers. Functionally, it is well described as a regulator of the actin cytoskeleton, but its role in gene regulation is less understood. Using primary mouse keratinocytes with a deletion of the RhoA gene, we have now been exploring how the loss of RhoA affects gene expression. Performing transcription factor reporter assays, we found a significantly decreased activity of a RAR luciferase reporter in RhoA-null keratinocytes. Inhibition of the RhoA effector ROCK in control cells reproduced this phenotype. ATRA and retinal, but not retinol increased RAR reporter activity of keratinocytes with impaired RhoA/ROCK signaling, suggesting that retinol metabolism is regulated by RhoA/ROCK signaling. Furthermore a significant percentage of known ATRA target genes displayed altered expression in RhoA-null keratinocytes. These data reveal an unexpected link between the cytoskeletal regulator RhoA and retinoid signaling and uncover a novel pathway by which RhoA regulates gene expression.

Keywords: ROCK; RhoA; retinoic acid; skin; stem cells.

Figure 1.

Decreased RAR reporter activity in RhoA-null keratinocytesCultured control (white bars) and RhoA-null keratinocytes (black bars) were transfected with luciferase reporters for the indicated transcription factors as described in “Materials and Methods.” Shown is the firefly luciferase activity of the reporters normalized to the renilla luciferase activity indicating transfection efficiency (n: 3-15/3-15; ***: p < 0,001).

Figure 2.

Decreased RAR reporter activity in RhoA-null keratinocytes is dependent on RhoA, ROCK, RARα. (A) Cultured control (white bars) and RhoA-null keratinocytes (black bars) were transfected with luciferase reporters for RAR and treated for 24 h with 10 μM ER50891, as indicated. Shown is the firefly luciferase activity of the reporters normalized to the renilla luciferase activity indicating transfection efficiency (n: 3; *: p < 0,05). (B) Left: Cultured control (white bars) and RhoA-null keratinocytes (black bars) were co-transfected with luciferase reporters for RAR and an EGFP-RhoA fusion protein or EGFP constructs, as indicated. Shown is the firefly luciferase activity of the reporters normalized to the renilla luciferase activity indicating transfection efficiency (n: 3; *: p < 0,05). Right: Representative blots showing efficient expression of EGFP-RhoA fusion protein and endogenous RhoA in control and RhoA-null keratinocytes. (C) Cultured control keratinocytes were transfected with luciferase reporters for RAR and incubated for 24 h with 25 μM of the ROCK inhibitor Y27632 (black bar) or left untreated (white bar). Shown is the firefly luciferase activity of the reporters normalized to the renilla luciferase activity indicating transfection efficiency (n: 15; ***: p < 0,001).

Figure 3.

Retinal and ATRA rescue reduced RAR-signaling activity in ROCK inhibitor treated keratinocytes. (A-C) Cultured control keratinocytes were transfected with luciferase reporters for RAR-mediated signaling and treated for 24 h with 25 μM Y27632, 10 μM retinol (A), 2 μM retinal (B), 1 μM ATRA (C), or left untreated, as indicated. Shown is the firefly luciferase activity of the reporters normalized to the renilla luciferase activity indicating transfection efficiency. All the samples were statistically compared to untreated cells. (n: 4-14; **: p < 0,01; ***: p < 0,001). (D-F) Cultured RhoA-null keratinocytes were transfected with luciferase reporters for RAR-mediated signaling and treated for 24 h with 25 μM Y27632, 10 μM retinol (D), 2 μM retinal (E), 1μM ATRA (F), or left untreated, as indicated. Shown is the firefly luciferase activity of the reporters normalized to the renilla luciferase activity indicating transfection efficiency. All the samples were statistically compared to untreated cells (n: 4-14; **: p < 0,01; ***: p < 0,001). (G) Treatment for 24 h with 2 μM retinal induced increased cell death in RhoA-null keratinocytes. Shown are representative pictures of keratinocytes in culture treated as indicated (size bar = 50mm).

Figure 4.

Gene ontology of upregulated genes in RhoA-ko keratinocytes in vitro Significantly enriched gene ontology categories of upregulated genes in RhoA-ko keratinocytes in vitro according to EASE Score, a modified Fisher Exact P-Value (p < 0.01).

Figure 5.

ROCK inhibition increases expression of retinoic acid dependent genes in keratinocytes. (A) qRT-PCR of RNA samples from cultured control and RhoA-null keratinocytes. Shown are fold inductions from RhoA-ko compared to control keratinocytes (n > 3; *: p < 0.05; **: p < 0,01; ***: p < 0,001). (B) Cultured RhoA-null keratinocytes were treated for 24 h with 1 μM ATRA. RNA was isolated and qRT-PCR was carried out for the indicated genes. Shown are fold inductions from treated keratinocytes compared to untreated keratinocytes. All the samples were statistically compared to untreated cells (n > 3; *: p < 0.05; **: p < 0,01; ***: p < 0,001). (C) qRT-PCR of RNA samples from cultured control and ROCK1/2-ko keratinocytes. Shown are fold inductions from ko compared to control keratinocytes (n > 3; *: p < 0.05; **: p < 0,01; ***: p < 0,001). (D) Representative blot showing efficient loss of ROCK1 and ROCK2 protein in ROCK1 and/or ROCK2-ko keratinocytes.

Figure 6.

α6^highCD34^high stem cells increased in the absence of RhoA. (A) Representative dot plot and quantification of FACS analysis for α6^highCD34^high cells in control and RhoA-ko freshly isolated keratinocytes from epidermis. (n: 4; *: p < 0.05). (B) Clonogenic assay of α6^highCD34^high sorted epidermal cells from control mice treated with 1 μM ATRA, 25 μM Y27632 or 10 μM ER50891 as indicated (n: 3 in triplicate; *: p < 0.05; ***: p < 0,001).

Figure 7.

RhoA/ROCK regulates RA signaling in primary keratinocytes RhoA/ROCK signaling regulates the oxidation of retinol to retinal.