Forkhead Box C1 Regulates Human Primary Keratinocyte Terminal Differentiation

Abstract

The epidermis serves as a critical protective barrier between the internal and external environment of the human body. Its remarkable barrier function is established through the keratinocyte (KC) terminal differentiation program. The transcription factors specifically regulating terminal differentiation remain largely unknown. Using a RNA-sequencing (RNA-seq) profiling approach, we found that forkhead box c 1 (FOXC1) was significantly up-regulated in human normal primary KC during the course of differentiation. This observation was validated in human normal primary KC from several different donors and human skin biopsies. Silencing FOXC1 in human normal primary KC undergoing differentiation led to significant down-regulation of late terminal differentiation genes markers including epidermal differentiation complex genes, keratinization genes, sphingolipid/ceramide metabolic process genes and epidermal specific cell-cell adhesion genes. We further demonstrated that FOXC1 works down-stream of ZNF750 and KLF4, and upstream of GRHL3. Thus, this study defines FOXC1 as a regulator specific for KC terminal differentiation and establishes its potential position in the genetic regulatory network.

Fig 1. FOXC1 is induced in differentiated keratinocytes.

A) mRNA levels of FOXC1 is increasing during the course of KC differentiation. Data presented as mean ±SEB. “undiff” is the abbreviation of undifferentiated KC. * p<0.05; ** p<0.01; *** p<0.001. The data are representative of four independent experiments. B) FOXC1 protein is increasing along with KC differentiation degree. The data represent one of the three independent experiments. C) Immunohistochemistry staining of FOXC1 protein expression in human epidermis, hair follicle and the differentiated inner hair root sheath. FOXC1 positive staining is presented as brown color in cell nuclear (Scale bar, 50 μm). The arrows point to examples of typical positive staining.

Fig 2. Silencing FOXC1 leads to deceased gene expression of KC differentiation markers.

A) mRNA expression of FLG, IVL and LOR in FOXC1 silenced differentiated KC and control cells evaluated by real-time PCR. Data represented as mean ±SEB. The data are representative of four independent experiments. B) Protein expression of FLG, IVL, LOR, DSC1 and Cyclin D in FOXC1 silenced differentiated KC and control cells evaluated by western -blot. The data represent one of the three independent experiments.

Fig 3. Over-expression of FOXC1 leads to increased gene expression of KC differentiation markers.

mRNA expression of FLG, IVL and LOR in FOXC1 over-expressed KC and control cells evaluated by real-time PCR. Data represented as mean ±SEB. The data are representative of three independent experiments.

Fig 4. Transcription profiles of FOXC1 silenced KC are significantly different from control cells upon differentiation.

A) A principle component assay shows separation of differentiated FOXC1 silenced KC from control cells. B) Heatmap shows the top 50 most differentially expressed genes in FOXC1 silenced undifferentiated and differentiated KC as compared to scrambled siRNA silenced undifferentiated and differentiated KC (FDR<0.01).

Fig 5. FOXC1 acts down-stream of ZNF750 and KLF4, and up-stream of GRHL3.

A) FOXC1 expression in ZNF750 and KLF4 silenced KC. B) ZNF750 expression in FOXC1 and KLF4 silenced KC. C) KLF4 expression in ZNF750 and FOXC1 silenced KC. D) GRHL3 expression in ZNF750, KLF4 and FOXC1 silenced KC. Gene expression was evaluated by real time PCR. Data represented as mean ±SEB. The data are representative of three independent experiments.

Fig 6. ZNF750 and KLF4 bind to the promoter region of FOXC1 gene.

A) ChIP assays were performed using Ca²⁺ -driven differentiated keratinocytes. 9 pairs of primers covering 2 kilobase region of the upstream of FOXC1 start codon. Increased binding activity was found in the regions of -873bp to -643bp and -663bp to -493bp up-stream of the start codon. Data represented as mean ±SEB. B) The Schematic diagram of FOXC1 promoter region. KLF4 and ZNF750 binding sites are indicated.