Role of the TGF-β signaling pathway in induced pluripotent stem cells reprogramming

To the Editor: The discovery of the induced pluripotent stem cells (iPSCs) by Takahashi and Yamanaka in 2006 was hailed as a major breakthrough in stem cell research. And a large number of experimental studies have proven that the transforming growth factor-β (TGF-β) signaling pathway is a key signaling pathway in iPSC reprogramming. The TGF-β signaling pathway can promote or inhibit iPSC reprogramming under different conditions [Figure 1]. This article describes the role of the TGF-β signaling pathway in iPSC reprogramming.Figure 1: The TGF-β signaling pathway can promote or inhibit iPSC reprogramming under different conditions. Akt: protein kinase B; BMP: Bone Morphogenetic Protein; Erk: extracellular signal-regulated kinase; GRB2: growth factor receptor-bound protein 2; iPSC: induced pluripotent stem cell; JNK: c-Jun N-terminal Kinase; Klf4: kruppel-like factor 4; LIMK: Lim-kinase; MEK: mitogen-activated protein kinase kinase; MEKK1: mitogen-activated protein kinase kinase kinase 1; MIS: Müllerian inhibiting substance; MKK:MAP kinase kinase; MLC: myosin light chain; MLK3: mixed lineage kinase 3; mTOR: mechanistic target of rapamycin; Oct4: octamer-binding transcription factor 4; PI3K: phosphatidylinositol 3-kinase; p38: p38 mitogen-activated protein kinase; Raf: rapidly accelerated fibrosarcoma; Ras: rat sarcoma; RhoA: ras homolog gene family member A; ROCK: Rho-associated protein kinase; ShcA: Src homology 2 domain-containing protein A; SOS: son of sevenless; Sox2: SRY-box transcription factor 2; S6K: S6 Kinase; TAK1: transforming growth factor beta-activated kinase 1; TGF-β: Transforming growth factor-β.According to homology, the TGF-β superfamily can be divided into three large subfamilies: various subtypes of TGF-β (β1, β2, β3), activin, and bone morphogenetic protein (BMP). The TGF-β family transmits signals through receptors, which can be divided into type I receptors (TβRI)/activin receptor-like kinase (ALK5), type II receptors (TβRII), and type III receptors (TβRIII) according to their structural and functional properties. TβRI and TβRII complex is required for TGF-β signal transduction. Upon binding to the ligand, TβRII protein kinase catalyzes the phosphorylation of serine and threonine residues in the guanidine specificity (GS) region of TβRI, resulting in the phosphorylation of a group of downstream signaling protein molecules called Smads in the cell. Smad plus general transcription factor (GTF), other transcription factors, or supplementary protein targets gene transcription. In addition, TGF-β/BMP cytokines can also activate non-Smad signaling molecules. For example, activated TβRI induces the formation of the Src homology 2 domain-containing protein A (ShcA)/growth factor receptor-bound protein 2 (GRB2)/son of sevenless (SOS) complex by phosphorylating ShcA and further activates extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) through the Rat sarcoma (Ras)-rapidly accelerated fibrosarcoma (Raf)-Mitogen-activated protein kinase kinase (MEK) pathway; the TGF-β receptor induces TNF receptor associated factor 6 (TRAF6) to catalyze transforming growth factor beta-activated kinase 1 (TAK1) to undergo K63-linked polyubiquitination, which then activates c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) via MAP kinase kinase (MKK). TGF-β can also induce its receptor to bind to p85, the regulatory subunit of phosphoinositide 3-kinase (PI3K), and activate PI3K-protein kinase B (Akt)-mechanistic target of rapamycin (mTOR) signaling for transcriptional regulation. Activated AKT also activates epithelial-mesenchymal transition (EMT) by inhibiting the transcriptional regulation of ribonucleoprotein E1. In addition, TGF-β affects the activity of other EMT-triggering signaling pathways, such as the Notch, Wnt, and integrin signaling pathways. Meanwhile, the TGF-β-induced non-Smad pathway can regulate Smad signaling. The TGF-β signaling pathway regulates cell differentiation. TGF-β1 has been shown to promote the transformation of human-induced pluripotent stem cells (hiPSCs) -derived neural stem cells (NSCs) into neurons and astrocytes in vitro. The optogenetic modulation of TGF-β signaling can also precisely control the differentiation of hiPSCs into the mesenchymal lineage.[1] It has also been shown that the hydrodynamic stress generated by shaking culture stimulates TGF-β signaling pathway in chondrogenically induced iPSCs (CI-iPSCs), promoting chondrogenic differentiation of mouse iPSCs in vitro. Inhibition of TGF-β can also regulate the cell differentiation ability. RepSox displaces SRY-box transcription factor 2 (Sox2) by inhibiting the ubiquitously expressed TGF-β signaling pathway in cultures containing stable intermediate cells captured by a partially reprogrammed state. This process, in turn, leads to sustained transcription of Nanog gene, resulting in reprogramming in the absence of Sox2.[2] Moreover, Misaki Yamashita et al[3] also found that inhibition of TGF-β could promote the differentiation of human iPSCs into iPSC-derived brain microvascular endothelial-like cells (iBMELCs) and increase the proportion of endothelial cells in differentiated cell population. It was found that inhibition of MEK, DNMT, and TGF-β was effective in inducing the differentiation of human iPSCs into enterocytes. Human iPSC-derived noncardiomyocyte cardiac cells (hiPSC-NMCCs) differentiate into myofibroblast-like cells when cultured with TGF-β or when transplanted into infarcted mouse hearts.[4] Gong et al[5] achieve directed differentiation of hiPSCs-derived neural crest cells (NCCs) by using chemically defined media containing glycogen synthesis kinase (GSK-3) inhibitors and TGF-β inhibitors. Indeed, TGF-β signaling has been shown to be essential for maintaining the pluripotency of human pluripotent stem cells (hPSCs). Single-cell RNA sequencing of iPSCs performed by Mabrouk et al[6] revealed that the TGF-β superfamily cytokines have important functions in pluripotency maintenance and cell differentiation during the early embryonic stages. In hPSC, the transcriptional regulator ZNF398 mediates pluripotency and epithelial properties downstream of TGF-β. Mechanistically, ZNF398 binds the active promoter and enhancer to SMAD3 and the histone acetyltransferase EP300, enabling TGF-β target transcription.[7] In the context of somatic reprogramming, inhibition of ZNF398 disrupts pluripotency and epithelial gene activation as well as colony formation.[7] The maintenance of pluripotency in iPSCs cultured in hESF 9 medium is absolutely dependent on the presence of TGF-β1. Activin and Nodal signaling play important roles in maintaining stem cell pluripotency associated with the TGF-β superfamily signaling networks. Upon PI3K activation, EMT/AKT establishes conditions where activin A/SMAD2/3 stimulate self-renewal in hiPSCs by activating target genes, including NANOG. Singh et al[8] found that PI3K/AKT inhibited RAF/MEK/ERK and typical Wnt signaling pathways, allowing SMAD2/3 to activate pluripotency-related target genes such as NANOG in the presence of a high level of PI3K in the undifferentiated state.[8] TGF-β signaling inhibits mesenchymal-epithelial transition (MET), an important early event in the transformation of fibroblasts into iPSCs. Inhibition of TGF-β signaling provides a possible mechanism to promote the generation of iPSCs. Small molecules targeting the MET machinery, such as the TGF-β receptor (SB431542), MEK (PD0325901), or ROCK (thiazovivin), alone or in combination, significantly increased the reprogramming efficiency. According to current studies, BMP signaling plays an important role in promoting early reprogramming: BMP7 significantly increased the percentage of iPSC reprogramming cell colonies induced by four OSKM genes (OCT4, KLF4, SOX2, and c-Myc) in mouse embryonic fibroblasts (MEFs).[9] Inhibiting TβRI kinase enhanced the efficiency and kinetics of OSKM-reprogrammed MEFs, while activation of the TGF-β pathway prevented reprogramming. Considering TβRI inhibitor treatment is most effective in the early stages of iPSC production, it is believed to exert a synergistic effect with reprogramming factors rather than promoting fibroblast transformation. Besides, it has been evidenced that TβRI inhibitor therapy can replace the solitary effect of C-MYC or SOX2, which suggests that TβRI acts by bypassing the programming factors C-MYC and SOX2 entirely. Ruetz et al[10] showed that constitutive activation of SMAD2/3 increases the efficiency of cell reprogramming in iPSCs thereby serving as a general factor that can enhance transcription factor-mediated reprogramming. Studies have shown that Math6 can counteract TGF-β signaling, affecting the initiating steps of cellular reprogramming, as well as the maintenance of pluripotency and early differentiation. However, there are still many limitations in the research on the specific mechanism of the TGF-β signaling pathway. For instance, the functions of the TGF-β signaling pathway remain elusive; the regulation of this pathway undergoes dynamic changes in response to different conditions; and there are still unidentified genes involved in its regulation as well as unknown potential for understanding their participation in modulating this pathway. In conclusion, in the process of exploring the reprogramming mechanism, it was found that the expression of TGF-β signaling pathway changed significantly. However, the specific regulatory mechanisms in the process of reprogramming remain to be further studied. Funding This work was supported by grants from the Key Projects in Yunnan Province (No. 202301AY070001-034) and the Project of the Yunnan Provincial Department of Education (No. 2023Y0827). Conflicts of interest None.