RACK1 Prevents the Premature Differentiation of Epidermal Progenitor Cells by Inhibiting IRF6 Expression

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TO THE EDITOR
To properly maintain barrier function, epidermal stem and progenitor cells, which reside in the basal layer of the skin, continually proliferate and differentiate to contribute to the outermost layers. Perturbations in the balance between stem cell self-renewal and differentiation leads to a variety of skin disorders (Blanpain and Fuchs, 2009). Thus, it is critical to understand the factors that govern basal layer cell fate decisions. We and others have identified IRF6, ZNF750, KLF4, and GRHL3 as transcription factors necessary to transition basal layer cells to a differentiated one (Oberbeck et al., 2019, Segre et al., 1999, Sen et al., 2012, Ting et al., 2005. However, less is known about how the levels of these transcription factors are regulated to prevent premature differentiation of stem and progenitor cells. To identify putative factors enriched in epidermal progenitor cells, we mined our previous gene expression profiling data comparing undifferentiated and differentiated primary human keratinocytes . Interestingly, the receptor for activated C kinase 1 also known as RACK1 (GNB2L1) was highly downregulated upon epidermal differentiation. RACK1 is a WD40 containing scaffolding protein that was originally identified as an anchoring protein for activated protein kinase C (Ron et al., 1994). As a scaffolding protein, it has been found to be associated with a large variety of proteins to affect a wide range of cellular processes (Li and Xie, 2015). RACK1 knockout mice are embryonic lethal at the gastrulation stage, which has precluded further examination of RACK1 in normal tissue homeostasis (Volta et al., 2013). Currently, it is unclear whether RACK1 has any role in stem cell fate decisions.
To answer this question, we use human skin as a model system. Staining of adult human skin showed that RACK1 is expressed primarily in the basal layer ( Figure 1a). Notably, the vast majority of RACK1 did not co-localize with keratin 1 (K1) which is expressed in the differentiated layers of the skin (Figure 1a). In primary human keratinocytes, RACK1 mRNA and protein levels are downregulated upon induction of differentiation with calcium ( Figure 1b). To test the function of RACK1, we knocked down RACK1 using two distinct shRNAs (RACK1i-A and RACK1i-B). Significant depletion of RACK1 as compared to scrambled shRNA controls (CTRi) was achieved on both the mRNA and protein levels ( Figure 1c). Loss of RACK1 expression resulted in the spontaneous upregulation of epidermal differentiation genes and inhibited cell proliferation without impacting apoptosis (Figure 1d-f). Together, these data suggest that the loss of RACK1 led the cells to prematurely differentiate and exit the cell cycle.
Next, we analyzed the gene expression profile of RACKi in comparison to CTRi keratinocytes grown in proliferation conditions. The loss of RACK1 resulted in the differential  Table S1).
In summary, we have found a basal layer enriched protein that is critical for preventing premature differentiation by inhibiting the expression of differentiation promoting transcription factors.

DATA AVAILABILITY
Datasets related to this article can be found at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE181265, hosted at the Gene Expression Omnibus (GEO): GSE181265.

Figure 1. RACK1 is required for maintaining epidermal progenitor cell function. (a)
Immunofluorescent staining of human skin with antibodies against RACK1 (red) and Keratin 1 (K1: green), a marker of the suprabasal layer. Merged imaged includes Hoechst 33342 staining of nuclei. n=3. Scale bar=10um. (b) RACK1 mRNA and protein expression in proliferating (-Ca +2 ) and differentiated (+Ca +2 day 3) primary human keratinocytes maintained in 2D culture. For RT-qPCR analysis of RACK1 mRNA levels, expression was normalized to L32. n=3. Data represented as mean ± SD. ** p<0.01 vs proliferating (-Ca +2 ) cells (unpaired t-test). For Western blot of RACK1 protein levels, -actin is shown as loading control. Representative image is shown and n=3. (c) Primary human keratinocytes were knocked down with control (CTRi) or RACK1 (RACK1i) shRNAs and the remaining RACK1 mRNA and protein levels were measured by RT-qPCR and Western blotting respectively. Two separate shRNAs (RACKi-A, RACK1i-B) targeting different regions of RACK1 were used. RACK1 mRNA expression was normalized to L32 levels. n=3. Data represented as mean ± SD. *** p<0.001 vs CTRi (unpaired t-test). For Western blot of RACK1 protein, -actin is shown as loading control. Representative image is shown and n=3. (d) RT-qPCR analysis of the relative mRNA expression of epidermal differentiation genes in CTRi and RACK1i cells. Cells were cultured in proliferation conditions. Expression of differentiation genes were normalized to L32 levels. n=3. Data represented as mean ± SD. * p<0.05 vs CTRi (unpaired t-test). (e) Proliferation assay of CTRi vs RACK1i cells. Plotted values represent the relative increase in fluorescence at each timepoint relative to starting point (day 0). n=3. Data represented as mean± SD. ** p<0.01 vs CTRi (1-way ANOVA). (f) Percent of apoptotic cells, comparing CTRi and RACKi cells, were analyzed by flow cytometry. Only cells positive for Annexin-V and negative for Propidium Iodide was considered as apoptotic. 6000 cells per measurement. n=3. Data represented as mean ± SD. ns= not significant.

RNA sequencing and analysis
Control (CTRi) and RACK1 (RACK1i) knockdown samples (transduction with retroviruses expressing shRNAs) were cultured in proliferation conditions for 7 days and harvested for RNA-Seq. Sequencing of extracted RNA was performed on the Illumina Hi Seq 2000, carried out by the Scripps Next Generation Sequencing Core Facility. Reads were aligned to the GENCODE v19 transcriptome hg19 using TopHat2 (Kim et al., 2013) with default settings. Normalization of reads and differential gene expression between samples was calculated by DESeq2 (Love et al., 2014) using Partek Flow (Partek Incorporated, http://www. partek.com/partek-genomics-suite). Enrichr (Kuleshov et al., 2016) was used to generate gene ontology (GO) terms for lists of differentially expressed genes. The statistical threshold of p≤0.05 and ≥1.5 fold change was used to create a gene list of differentially upregulated and downregulated genes in comparison to control samples. Partek Flow was used to generate heatmaps representing relative expression of genes. ARCHS4  was used to identify co-expressing transcription factors from the list of differentially upregulated genes identified in RACK1i keratinocytes .

Statistical analysis
All data are presented as mean±s.d. and analyzed using Graphpad Prism. Statistical significance was assessed using two-way ANOVA when comparing multiple treatments between two or more groups, one-way ANOVA when comparing multiple treatments within a single group or an unpaired t-test when comparing two treatments.