A thyroid hormone receptor/KLF9 axis in human hepatocytes and pluripotent stem cells

Abstract

Biological processes require close cooperation of multiple transcription factors that integrate different signals. Thyroid hormone receptors (TRs) induce Krüppel-like factor 9 (KLF9) to regulate neurogenesis. Here, we show that triiodothyronine (T3) also works through TR to induce KLF9 in HepG2 liver cells, mouse liver, and mouse and human primary hepatocytes and sought to understand TR/KLF9 network function in the hepatocyte lineage and stem cells. Knockdown experiments reveal that KLF9 regulates hundreds of HepG2 target genes and modulates T3 response. Together, T3 and KLF9 target genes influence pathways implicated in stem cell self-renewal and differentiation, including Notch signaling, and we verify that T3 and KLF9 cooperate to regulate key Notch pathway genes and work independently to regulate others. T3 also induces KLF9 in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC) and this effect persists during differentiation to definitive endoderm and hiPSC-derived hepatocytes. Microarray analysis reveals that T3 regulates hundreds of hESC and hiPSC target genes that cluster into many of the same pathways implicated in TR and KLF9 regulation in HepG2 cells. KLF9 knockdown confirms that TR and KLF9 cooperate to regulate Notch pathway genes in hESC and hiPSC, albeit in a partly cell-specific manner. Broader analysis of T3 responsive hESC/hiPSC genes suggests that TRs regulate multiple early steps in ESC differentiation. We propose that TRs cooperate with KLF9 to regulate hepatocyte proliferation and differentiation and early stages of organogenesis and that TRs exert widespread and important influences on ESC biology.

Keywords: Human embryonic stem cell; Induced pluripotent stem cell; Krüppel-like factor 9; Notch; Thyroid receptor.

Figure 1.

T3 induces KLF9 in HepG2 cells and in hepatocytes. (A): Parental HepG2, HepG2-TRβ, and HepG2-TRα cells were treated with 100 nM T3 for the indicated times and KLF9 mRNA levels were determined by quantitative real-time PCR (qPCR). (B–D): Expression of KLF9 after T3 treatment was assessed by qPCR in mice liver tissue (B), in isolated mice hepatocytes treated with increasing concentrations of T3 (C), and human hepatocytes isolated from neonatal and adult livers (D).

Figure 2.

TR/KLF9 axis regulates multiple genes in HepG2 cells. (A): Differential gene regulation in HepG2-TRb cells after KLF9 silencing revealed by microarray analysis. Microarray data obtained from human Illumina HT-12_v4 gene chips from control versus KLF9 knockdown were analyzed using Limma package within R. Effects determined to be significant when more than or equal to twofold with an adjusted p-value ≤ .05. (B): Effects of KLF9 knockdown confirmed at representatives of both classes of gene by quantitative real-time PCR. (C): Differential gene regulation by T3 in control and siKLF9 cells revealed by microarray analysis. (D–H): Cells were treated with 100 nM T3 and qRT-PCR was performed to verify patterns of KLF9-dependency of T3 response. Data are represented as mean ± SD. Microarray data are deposited in the Gene Expression Omnibus; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=sxkvckgwhxmfdgx&acc=GSE54699; accession number GSE54699.

Figure 3.

TR/KLF9 axis and the Notch pathway. (A–C): Cells were treated with 100 nM T3 and qRT-PCR was performed to verify TR/KLF9-dependency of identified Notch pathway genes. (D): Network of interactions among Notch pathway KLF9 targets, as retrieved by the GeneMania. Circles represent genes and connecting lines represent interactions between genes. GeneMania retrieved known and predicted interactions between these genes and added extra genes (gray circles) that are strongly connected to query genes. (E–G): Quantitative real-time PCR verification of genes identified by GeneMania as part of TR/KLF9-Notch network. All data are represented as mean ± SD.

Figure 4.

TR/KLF9 axis is active in embryonic stem cells, definitive endoderm, and human induced pluripotent stem cell (hiPSC)-derived hepatocytes. (A–C): Quantitative real-time PCR (qPCR) analysis of KLF9 expression levels 2/1T3 in BJ fibroblasts, iKCL004, iKCL011, and KCL034 cells (A), endoderm differentiated from iKCL004, iKCL011, and KCL034 (B), and terminally differentiated hiPSC-derived hepatocytes (C). KLF9 mRNA levels were expressed as fold change. All data are represented as mean ± SD. (D–H): TR/KLF9 axis is involved in regulation of Notch signaling in hiPSC and human embryonic stem cell. (D): Western blot for KLF9 from cell lysates from iKCL004, iKCL011, and KCL034 transfected with Ctrl or KLF9 siRNA. (E–H): iKCL004, iKCL011, and KCL034 cells transfected with Ctrl or KLF9 siRNA were treated with 100 nM T3 for 18 hours. KLF9, HES4, PSEN2, and HES5 mRNA levels were determined by qPCR. The data are presented as fold change of mRNA levels in Ctrl nontreated samples. All data are represented as mean ± SD.

Figure 5.

Analysis of representative gene expression in KCL034 and iKCL004 cells. (A): T3-induced expression of HHEX, HAND1, and POU3F1 in iKCL004 and KCL034. (B, C): Nanog display T3-dependent reduction in expression levels in KCL034 and iKCL004 as verified by qPCR (B) and confirmed by immunostaining in KCL034 cells (C). (D): A model for TR/KLF9 action. TR activates transcription of KLF9 and both transcription factors modulate each other’s activity in multiple pathways leading to cell-specific responses to different signals. Abbreviation: TR, thyroid hormone receptor.