Keratin 79 is a PPARA target that is highly expressed by liver damage

Keratins are key structural proteins found in skin and other epithelial tissues. Keratins also protect epithelial cells from damage or stress. Fifty-four human keratins were identified and classified into two families, type I and type II. Accumulating studies showed that keratin expression is highly tissue-specific and used as a diagnostic marker for human diseases. Notably, keratin 79 (KRT79) is type II cytokeratin that was identified as regulator of hair canal morphogenesis and regeneration in skin, but its role in liver remains unclear. KRT79 is undetectable in normal mouse but its expression is significantly increased by the PPARA agonist WY-14643 and fenofibrate, and completely abolished in Ppara-null mice. The Krt79 gene has functional PPARA binding element between exon 1 and exon 2. Hepatic Krt79 is regulated by HNF4A and HER2. Moreover, hepatic KRT79 is also significantly elevated by fasting- and high-fat diet-induced stress, and these increases are completely abolished in Ppara-null mice. These findings suggest that hepatic KRT79 is controlled by PPARA and is highly associated with liver damage. Thus, KRT79 may be considered as a diagnostic marker for human liver diseases.


Introduction
Keratins are the intermediate filament-forming protein in epithelial cells.The keratin family consists of 54 proteins in two families (type I, acidic, and II, neutral or basic), each being expressed in during differentiation [1].Keratin proteins consist of simple, barrier and structural forms [2].Keratins are normally expressed in a cell type-, differentiation-and functional status-dependent manner.Epithelial cancers largely maintain the characteristics of keratin expression associated with their respective cell type of origin [3].Keratins have long been recognized as diagnostic markers in tumor pathology [3].Notably, keratin 79 (KRT79) is type II keratins identified in a novel population of migratory epithelial cells that initiate hair canal morphogenesis and regeneration [4].KRT79 has an important role in the maintenance of sebaceous gland stem cells in the skin [5].Loss of KRT79 caused abnormal sebaceous glands and eyelid meibomian glands [5].Proteomic analysis to identify the mechanism of the anticancer properties of flavonoids from citrus platymamma showed that KRT79 expression is a possible modulator of cell differentiation and cell motility, is down-regulated by citrus platymamma in A549 lung cancer cells [6].A recent study showed that hepatic KRT23 is controlled by peroxisome proliferator-activated receptor α (PPARA) and the proto-oncogene MYC that are known to promote hepatocyte proliferation [7].In the present study, basal expression levels of hepatic KRT79 was undetectable in mouse hepatocytes.but was highly induced by WY-14643 and fenofibrate, agonistic ligands for PPARA and completely abolished in Ppara-null mice.PPARA directly binds to the Krt79 gene.Furthermore, PPARA-mediated liver stress by fasting and high-fat diet significantly induced hepatic KRT79 expression in mice and it was not found in Ppara-null mice.KRT79 is encoded by a PPARA target gene and is highly elevated in by liver damage.

Primary hepatocyte isolation
previously reported [8] and seeded on collagen-coated 12-well plates (Becton Dickinson and Company) at a density of 2 × 10 5 cells in Williams' Medium E (Thermo-Fisher Scientific, Waltham, MA) supplemented with 5% FBS and penicillin/streptomycin/amphotericin B solution (Gemini Bio-products).The cells were harvested and subjected to qRT-PCR for mRNA quantification.

Immunohistochemistry of KRT79
Immunohistochemical staining was performed on formalin-fixed paraffin embedded sections from human or mouse liver.For immunohistochemical staining, sections were deparaffinized followed by antigen retrieval with sodium citrate buffer for 2 min at 95 °C and then placed at room temperature (RT) for 30 min.Sections were blocked using 5% BSA for 30 min followed by incubation with 10% goat serum for 30 min at RT.After blocking, sections were incubated with KRT79 (Thermo-Fisher Scientific; PA5-46517) primary antibody in a humidified chamber overnight at 4 °C.Following primary antibody incubation, sections were washed and incubated with secondary antibodies for 30 min (Gene Tech Company; GK600505).Following secondary antibody incubation, the sections were washed, and DAB substrate added (Vector Laboratories, Burlingame, CA) and developed by checking for staining under a microscope.The sections were then washed and counterstained with hematoxylin for 30 s.Sections were then washed with tap water, dehydrated, and mounted.All imaging was performed using a Keyence BZ-X700 series all-in-one microscope with 20x and 40x objectives (200x magnifications) (Keyence, Osaka, Japan).

Luciferase reporter assays
For luciferase assays, pSG5-PPARA (mouse) and pSG5-RXRA (mouse) were used for transcription factor expression [11].Custom GeneBlocks (IDT DNA, Coralville, IA) were synthesized containing the predicted PPRE sites on Krt79 gene.GeneBlocks were digested and purified using a Qiagen PCR Purification Kit (Qiagen, Valencia, CA), and cloned into the pGL4.11for PPRE constructs (Promega, Madison, WI) using a BioRad Quick Ligation Kit (BioRad, Hercules, CA).Prior to performing assays, all constructs were confirmed by Sanger sequencing at the NCI Center for Cancer Research Genomics Core.The phRL-TK renilla luciferase construct was used as a control to normalize for transfection efficiency.Primary hepatocytes were seeded into 12-well plates (4 × 10 4 cells/ well).PPRE reporter constructs were co-transfected into hepatocytes with PPARA and RXR expression.vectors.PPRE-luc plasmid containing an Acox1 peroxisome proliferator regulatory element (PPRE) site was used as a positive control [12].Empty pGL4.11plasmid was used as negative controls.Plasmids were transfected using Lipofectamine 3000 Reagent (Thermo-Fisher Scientific).Luciferase activities were measured and plotted relative to lysate protein concentrations using the Promega Dual Luciferase Reporter (DLR, Promega) assays according to the manufacturer's protocol.Measurements were taken on a Veritas microplate luminometer (Turner Biosystems, Sunnyvale, CA).

Mouse models
Ppara wild-type (Ppara −/− ) male mice and conventional Ppara −/− male mice used in this study were described [13].For WY-14643 studies, mice were provided a grain-based control diet or matched diet containing 0.1% WY-14643 for 2 days.For monitoring the time dependence of Krt79 gene responses, WY-14643 was dissolved in 1% carboxymethyl cellulose (CMC) solution and orally administered (50 mg/kg in 200 μl) for the indicated time points.Hepatocyte nuclear factor 4α (Hnf4a) wild-type male mice and hepatocytespecific Hnf4a-null (Hnf4a ΔHep ) male mice used in this study were described [14].For estrogen receptor activation studies, 2 mg of tamoxifen was intraperitoneally injected.For fasting studies, food was removed for 24 h starting shortly after the onset of the light cycle and endpoints collected at the same time the following day.For the HFD feeding experiment, mice were fed a HFD (60% kcal from fat) for 16 weeks.Mice were housed in light and temperature-controlled rooms and were provided with water and pelleted chow ad libitum.Mice were then killed, and tissue samples harvested, snap frozen in liquid nigrogen and stored at −80 °C for further analysis.All animal experiments were performed in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care international guidelines and approved by the National Cancer Institute Animal Care and Use Committee.

Computational and statistical analysis
All results are expressed as means ± SD.Significance was determined by t-test or one-way ANOVA with Bonferroni correction using Prism 7.0 software (GraphPad Software, La Jolla, CA).A P value less than 0.05 was considered significant and statistical significance is indicated in the figure legends.

KRT79 response is PPARA-dependent and liver specific
Basal expression levels of Krt79 mRNA were highest in brown adipose, lung and muscle but very low in liver (Fig. 1A).KRT23 is type I cytokeratin also highly induced by ligand activation of PPARA [7].Induction of Krt79 mRNA by WY-14643 was liver specific and not observed in Ppara −/− mice, indicating that their expression is PPARA dependent (Fig. 1B).Krt79 mRNA was rapidly induced with maximum expression seen at 12 h after ligand administration (Fig. 1C).Fenofibrate treatment increased Krt79 mRNA expression and not observed in Ppara −/− mice (Fig. 1D).In primary hepatocytes, Krt79 mRNA was not increased by the PPARA antagonist GW6741 which also block its induction by WY-14632 (Fig. 1E).KRT79 staining was primarily cytosolic and was increased significantly in response to WY-14643 treatment when compared with controls; staining was markedly decreased in Ppara −/− mice (Fig. 2F).These data show that Krt79 mRNA expression in response to PPARA activation was liver specific, indicating a potential role for these proteins in hepatocyte proliferation.

PPARA directly regulates KRT79 by binding to promoter response elements
Genomatix MatInspector (Genomatix, Munchen, Germany) was used to bioinformatically identify potential PPREs on the Krt79 gene.Four PPRE sites were identified in the intron between Krt79 exon 1 and 2 (Fig. 2A).Inserts (A-D) containing the predicted PPRE sites were synthesized and cloned into the pGL4.11reporter and luciferase assays performed to confirm direct PPARA binding on the Krt79 gene.Another PPRE-luc construct containing an Acox1 PPRE repeat was used as a positive control and empty pGL4.11plasmid was used as a negative control.Luciferase activity was significantly elevated in primary hepatocytes transfected with the pGL4.11-Dconstruct which contained a single PPRE site, indicating direct regulation by PPARA (Fig. 2B).To check whether exon 1 is amplified after PPARA binding, exon specific primers were designed and subjected to mRNA quantification.Krt79 exon 1 is not transcribed in mouse liver after WY-14643 treatment (Fig. 2C).In Hepa-1 cells, Krt79 exon 1 RNA transcript level was very low compared to exon 2 and exon 4 RNA transcripts (Fig. 2D).Together these data indicate that activation of PPARA regulates Krt79 transcription primarily through direct binding to a PPRE sites found in the intron between exons 1 and 2.

KRT79 expression is affected by hepatocyte nuclear factor 4α and estrogen receptor
Bioinformatical analysis by Genomatix MatInspector (Genomatix, Munchen, Germany) revealed that the Krt79 gene has hepatocyte nuclear factor 4 α (HNF4A) and estrogen receptor (ER) binding elements.To identify whether hepatic Krt79 expression is affected by HNF4A and ER, Hnf4a ΔHep mice were treated WY-14643, and the ER antagonist tamoxifen was administered to wild-type mice.Hepatic Hnf4a mRNA expression was decreased in Hnf4a ΔHep mice treated with and without WY-14643 (Fig. 3A).However, WY-14643induced hepatic Krt79 mRNA expression was significantly inhibited in Hnf4a ΔHep mice treated with WY-14643 (Fig. 3B).Tamoxifen is breast cancer drug that competitively inhibits activation of the ER [15].Tamoxifen treatment induced hepatic Krt79 expression (Fig. 3C).Interestingly, hepatic Krt79 expression was significantly increased in combination with WY-14643 and tamoxifen (Fig. 3C).These data suggest that hepatic Krt79 expression is regulated by HNF4A and ER (Fig. 3).

KRT79 expression is highly elevated in liver damage
Recent studies showed that keratins are histological cancer diagnostic markers, particularly epithelial cell-derived cancers [16,17].KRT23 is PPARA dependent and also highly expressed in human liver cancer [7].To determine whether KRT79 is associated with liver disease, fasting and high-fat diet-induced liver disease models were analyzed for Krt79 expression.Krt79 mRNA expression was significantly increased in liver of fasted and highfat diet treated mice; it was not elevated in liver of Ppara −/− mice under these treatments (Fig. 4A-B).Basal expression levels of Krt79 mRNA is highly elevated in Hepa-1 cells compared to primary hepatocyte (Fig. 4C).These data indicated that KRT79 is highly associated with liver damage.

Discussion
Keratins are expressed in all type of epithelial tissues and play an important role in epithelial cell protection from stressors [18].Keratins are conventionally thought of as structural proteins, but more recently, several studies have revealed that they also play active roles in cell signaling pathways [19,20].This is first study to investigate the role of KRT79 in liver.Krt79 mRNA was abundant in brown adipose, lung, and muscle tissues.Basal expression of hepatic Krt79 mRNA was undetectable in livers of untreated wild-type mice.However, the PPARA agonist WY-14643 significantly induced Krt79 mRNA and KRT79 positive cells in livers of wild-type mice.Krt79 mRNA was unchanged in Ppara −/− mice treated with a PPARA agonist, indicating that Krt79 induction is PPARA dependent.Krt79 is robustly increased beginning 3 h after activation of PPARA by WY-14643.Hepatic Krt79 mRNA was also induced by fenofibrate in mice and by WY-14643 in primary hepatocyte cultures.These data revealed that hepatic Krt79 is PPARA target gene.
PPARA ChIP-seq analysis revealed that the Krt79 gene has potential PPARA binding sites (GSE61817).The underlying PPARA regulatory interactions controlling activation of the Krt79 promoter were investigated.PPARA binding sites were functionally identified in the intron between exons 1 and 2 of the Krt79 gene as revealed by luciferase reporter gene assays.To determine whether the expression of exon 1 RNA is increased after PPARA binding, qRT-PCR was perfomed on exons 1, 2, and 4 derived RNAs.The results showed that Krt79 exon 1 was not transcribed in WY-14643 treated mouse liver.Krt79 exon 1 transcripts were significantly lower than exon 2 and 4 transcripts in Hepa-1 cells.This result suggest that Krt79 exon 1 may act as negative regulator of Krt79.Thus, hepatic Krt79 is a direct PPARA target gene in WY-14643-stimulated mouse liver.Bioinformatical analysis revealed that the Krt79 gene has multiple transcription factor binding elements including those for HNF4A and ER.WY-14643-induced hepatic Krt79 was significantly inhibited in Hnf4a ΔHep mice treated with WY-14643.Tamoxifen is an anticancer and chemoprevention drug for ER-positive breast cancer, acting as an ER antagonist [15,21].Surprisingly hepatic Krt79 mRNA was also increased by tamoxifen.
Notably, treatment with WY-14643 and tamoxifen caused significantly increased hepatic Krt79 mRNA expression compared to treatment with WY-14643 or tamoxifen alone.These data suggested that HNF4α and ER can regulate hepatic Krt79 when Krt79 is activated by PPARA.
Fasting and high fat diet-induced pathological liver stress result in PPARA activation by endogenous ligands [22,23].Fasting and high fat diet also induced hepatic Krt79 mRNA expression, that was not found in liver of Ppara −/− mice.Furthermore, basal expression level of Krt79 in Hepa-1 cells originated mouse hepatoma was significantly elevated compared to primary hepatocytes.These data indicated that hepatic KRT79 is highly associated with liver damage.Taken together, these results demonstrate that hepatic Krt79 is a PPARA target gene and that KRT79 can be used as a biochemical or histological diagnostic biomarker for human liver diseases.