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Objective: The epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells (RTECs) plays a crucial role in renal interstitial fibrosis and inflammation, which are key components of chronic kidney disease (CKD). Alantolactone, a selective inhibitor of signal transducer and activator of transcription 3 (STAT3), is used in Chinese herbal medicine. Despite its use, the effects of alnatolactone on EMT of RTECs has not been fully elucidated. Methods: In this study, we investigated the potential of alantolactone to EMT in vivo and in vitro. Our experiments were performed using a unilateral ureteral obstruction (UUO) models and HK-2 cells, RTECs, treated with transforming growth factor (TGF-ß). Results: Alantolactone decreased tubular injury and reduced the expression of vimentin, a key EMT marker, while increasing E-cadherin expression in UUO kidneys. Similarly, in RTECs, alantolactone inhibited TGF-ß-induced EMT and its markers. Furthermore, alantolactone attenuated UUO- and TGF-ß-induced STAT3 phosphorylation both in vivo and in vitro, and inhibited the expression of TWIST, an EMT transcription factor, in both models. Conclusion: Alantolactone improves EMT in RTECs by inhibiting STAT3 phosphorylation and Twist expression, suggesting its potential as a therapeutic agent for kidney fibrosis.

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Pancreatic ductal adenocarcinoma (PDAC) presents significant oncological challenges due to its aggressive nature and poor prognosis. The tumor microenvironment (TME) plays a critical role in progression and treatment resistance. Non-neoplastic cells, such as cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs), contribute to tumor growth, angiogenesis, and immune evasion. Although immune cells infiltrate TME, tumor cells evade immune responses by secreting chemokines and expressing immune checkpoint inhibitors (ICIs). Vascular components, like endothelial cells and pericytes, stimulate angiogenesis to support tumor growth, while adipocytes secrete factors that promote cell growth, invasion, and treatment resistance. Additionally, perineural invasion, a characteristic feature of PDAC, contributes to local recurrence and poor prognosis. Moreover, key signaling pathways including Kirsten rat sarcoma viral oncogene (KRAS), transforming growth factor beta (TGF-ß), Notch, hypoxia-inducible factor (HIF), and Wnt/ß-catenin drive tumor progression and resistance. Targeting the TME is crucial for developing effective therapies, including strategies like inhibiting CAFs, modulating immune response, disrupting angiogenesis, and blocking neural cell interactions. A recent multi-omic approach has identified signature genes associated with anoikis resistance, which could serve as prognostic biomarkers and targets for personalized therapy.

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BACKGROUND: The intestinal epithelium performs essential physiological functions, such as nutrient absorption, and acts as a barrier to prevent the entry of harmful substances. Mycotoxins are prevalent contaminants found in animal feed that exert harmful effects on the health of livestock. Zearalenone (ZEA) is produced by the Fusarium genus and induces gastrointestinal dysfunction and disrupts the health and immune system of animals. Here, we evaluated the molecular mechanisms that regulate the effects of ZEA on the porcine intestinal epithelium. RESULTS: Treatment of IPEC-J2 cells with ZEA decreased the expression of E-cadherin and increased the expression of Snai1 and Vimentin, which induced Snail1-mediated epithelial-to-mesenchymal transition (EMT). In addition, ZEA induces Snail-mediated EMT through the activation of TGF-ß signaling. The treatment of IPEC-J2 cells with atractylenolide III, which were exposed to ZEA, alleviated EMT. CONCLUSIONS: Our findings provide insights into the molecular mechanisms of ZEA toxicity in porcine intestinal epithelial cells and ways to mitigate it.

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Bisphenol A (BPA) and its analogues (BPAF, BPS) are ubiquitous environmental contaminants used as plastic additives in various daily life products, with many concerns on their role as environmental estrogens. Uterine leiomyomas (fibroids) are highly prevalent gynecologic tumors with progressive fibrosis. Fibroids are hormone-responsive and may be the target of environmental estrogens. However, the effects of BPA, BPAF, and BPS exposure on uterine fibrosis are largely unknown. Here, we evaluated fibrosis and the crucial role of TGF-beta signaling in human fibroid tumors, the profibrotic effects of BPA, BPAF or BPS in a human 3D uterine leiomyoma (ht-UtLM) in vitro model, and the long-term outcomes of BPAF exposure in rat uterus. In 3D ht-UtLM spheroids, BPA, BPAF, and BPS all promoted cell proliferation and fibrosis by increasing the production of extracellular matrices. Further mechanistic analysis showed the profibrotic effects were induced by TGF-beta signaling activation mainly through SMAD2/3 pathway and crosstalk with multiple non-SMAD pathways. Furthermore, the profibrotic effects of BPAF were supported by observation of uterine fibrosis in vivo in rats following long-term BPAF exposure. Overall, the 3D ht-UtLM spheroid can be an important model for investigating environment-induced fibrosis in uterine fibroids. BPA and its analogues can induce fibrosis via TGF-beta signaling.

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BACKGROUND: The activation of TGF-ß pathway can facilitate tumorigenesis. Understanding the TGF-related genes (TRGs) in oral cancer and determining their prognostic value is of utmost importance. METHODS: The TRGs were selected to develop a prognostic model based on lasso regression. Oral cancer patients were classified into high-risk and low-risk groups based on the risk model. Subsequently, multivariate COX regression was employed to identify the prognostic marker. Additionally, the expression of SMURF2 was validated using quantitative real-time polymerase chain reaction (qRT-PCR) and the Human Protein Atlas (HPA) database. To investigate the relationship between SMURF2 expression and immune cell infiltrations, we conducted single-sample Gene Set Enrichment Analysis (ssGSEA) analyses. RESULTS: We identified 16 differentially expressed TRGs in oral cancer, all of which showed upregulation. From these, we selected eight TRGs as prognostic signatures. Furthermore, the high-risk group demonstrated lower infiltration levels of immune cells, immune score, and higher tumor purity. Interestingly, we also found that SMURF2 serves as an independent prognostic biomarker. SMURF2 was upregulated in oral cancer, as confirmed by public databases and qRT-PCR analysis. Importantly, our results indicate a close association between SMURF2 expression and the immune microenvironment. CONCLUSION: The 8-TRG signature prognosis model that we constructed has the ability to predict the survival rate and immune activity of oral cancer patients. SMURF2 could be effective in recognizing prognosis and evaluating immune efficacy for oral cancer.

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Pulmonary arterial hypertension associated with schistosomiasis (SchPAH) and pulmonary arterial hypertension associated with portal hypertension (PoPAH) are lung diseases that develop in the presence of liver diseases. However, mechanistic pathways by which the underlying liver conditions and other drivers contribute to the development and progression of pulmonary arterial hypertension (PAH) are unclear for both etiologies. In turn, these unknowns limit certainty of strategies to prevent, diagnose, and reverse the resultant PAH. Here we consider specific mechanisms that contribute to SchPAH and PoPAH, identifying those that may be shared and those that appear to be unique to each etiology, in the hope that this exploration will both highlight known causal drivers and identify knowledge gaps appropriate for future research. Overall, the key pathophysiologic differences that we identify between SchPAH and PoPAH suggest that they are not variants of a single condition.

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Pericyte dysfunction and loss contribute substantially to the destabilization and rupture of atherosclerotic plaques. Protocatechuic aldehyde (PCAD), a natural polyphenol, exerts anti-atherosclerotic effects. However, the effects and mechanisms of this polyphenol on pericyte recruitment, coverage, and pericyte function remain unknown. We here treated apolipoprotein E-deficient mice having high-fat diet-induced atherosclerosis with PCAD. PCAD achieved therapeutic effects similar to rosuvastatin in lowering lipid levels and thus preventing atherosclerosis progression. With PCAD administration, plaque phenotype exhibited higher stability with markedly reduced lesion vulnerability, which is characterized by reduced lipid content and macrophage accumulation, and a consequent increase in collagen deposition. PCAD therapy increased pericyte coverage in the plaques, reduced VEGF-A production, and inhibited intraplaque neovascularization. PCAD promoted pericyte proliferation, adhesion, and migration to mitigate ox-LDL-induced pericyte dysfunction, which thus maintained the capillary network structure and stability. Furthermore, TGFBR1 silencing partially reversed the protective effect exerted by PCAD on human microvascular pericytes. PCAD increased pericyte coverage and impeded ox-LDL-induced damages through TGF-ß1/TGFBR1/Smad2/3 signaling. All these novel findings indicated that PCAD increases pericyte coverage and alleviates pericyte damage to improve the stability of atherosclerotic plaques, which is accomplished by regulating TGF-ß1/TGFBR1/Smad2/3 signaling in pericytes.

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Background: TGF-beta signaling is a key regulator of immunity and multiple cellular behaviors in cancer. However, the prognostic and therapeutic role of TGF-beta signaling-related genes in ovarian cancer (OV) remains unexplored. Methods: Data of OV used in the current study were sourced from TCGA and GEO databases. Consensus clustering was applied to classify OV patients into different clusters using TGF-beta signaling-related genes. Differentially expressed genes (DEGs) between different clusters were screened by the "limma" R package. Prognostic genes were screened from DEGs by univariate Cox regression, followed by the construction of the TGF-beta signaling-related score. The prognostic value of TGF-beta signaling-related score was evaluated in both training and testing OV cohorts. Moreover, the immune status, GSEA and therapeutic response between low- and high-score groups were performed to further reveal the potential mechanisms. Results: By consensus clustering, OV patients were classified into two clusters with different tumor immune environments. After differential expression and univariate Cox regression analyses, GMPR, PIEZO1, EMP1, CXCL13, GADD45B, SORCS2, FOSL2, PODN, LYNX1 and SLC38A5 were selected as prognostic genes. Using PCA algorithm, the TGF-beta signaling-related score of OV patients was calculated based on prognostic genes. Then OV patients were divided into low- and high-TGF-beta signaling-related score groups. We observed that the two score groups had significantly different survivals, tumor immune environments and expressions of immune checkpoints. In addition, GSEA results showed that immune-related pathways and biological processes, like chemokine signaling pathway, TNF signaling pathway and T cell migration were significantly enriched in the low-score group. Moreover, patients in the low- and high-score groups had remarkably different sensitivity to chemo- and immunotherapy. Conclusion: For the first time, our study identified ten prognostic genes associated with TGF-beta signaling, constructed a prognostic TGF-beta signaling-related score and investigated the effect of TGF-beta signaling-related score on OV immunity and therapy. These findings may enrich our knowledge of the TGF-beta signaling in OV prognosis and help to improve the prognosis prediction and treatment strategies in OV.

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The endothelium is a mechanosensitive organ whose pleiotropic actions regulate vessel structure to adjust tissue perfusion. To do so, it possesses ion channels, receptor complexes, and signaling pathways responding to blood flow, whose activation will either maintain vascular integrity and quiescence or, on the contrary, remodel the vessel's structure in both health and disease. Recent studies have demonstrated the crucial role of endothelial inflammation, endothelial to mesenchymal transition (EndMT), and perturbed hemodynamics in the progression of pulmonary arterial hypertension and essential hypertension. These two distinct diseases share some common mechanistic cues, pointing towards new potential therapeutic approaches to treat them. In this review, we summarize these common mechanisms to map future drug development strategies targeting flow sensing mechanisms and vascular remodeling.

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Molecular mechanisms underlying cognitive deficits in Huntington's disease (HD), a striatal neurodegenerative disorder, are unknown. Here, we generated ChIPseq, 4Cseq and RNAseq data on striatal tissue of HD and control mice during striatum-dependent egocentric memory process. Multi-omics analyses showed altered activity-dependent epigenetic gene reprogramming of neuronal and glial genes regulating striatal plasticity in HD mice, which correlated with memory deficit. First, our data reveal that spatial chromatin re-organization and transcriptional induction of BDNF-related markers, regulating neuronal plasticity, were reduced since memory acquisition in the striatum of HD mice. Second, our data show that epigenetic memory implicating H3K9 acetylation, which established during late phase of memory process (e.g. during consolidation/recall) and contributed to glia-mediated, TGFß-dependent plasticity, was compromised in HD mouse striatum. Specifically, memory-dependent regulation of H3K9 acetylation was impaired at genes controlling extracellular matrix and myelination. Our study investigating the interplay between epigenetics and memory identifies H3K9 acetylation and TGFß signaling as new targets of striatal plasticity, which might offer innovative leads to improve HD.

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Background: Thyroid hormones play a significant role in bone development and maintenance, with triiodothyronine (T3) particularly being an important modulator of osteoblast differentiation, proliferation, and maintenance. However, details of the biological processes (BPs) and molecular pathways affected by T3 in osteoblasts remain unclear. Methods: To address this issue, primary cultures of human adipose-derived mesenchymal stem cells were subjected to our previously established osteoinduction protocol, and the resultant osteoblast-like cells were treated with 1 nm or 10 nm T3 for 72 h. RNA sequencing (RNA-Seq) was performed using the Illumina platform, and differentially expressed genes (DEGs) were identified from the raw data using Kallisto and DESeq2. Enrichment analysis of DEGs was performed against the Gene Ontology Consortium database for BP terms using the R package clusterProfiler and protein network analysis by STRING. Results: Approximately 16,300 genes were analyzed by RNA-Seq, with 343 DEGs regulated in the 1 nm T3 group and 467 upregulated in the 10 nm T3 group. Several independent BP terms related to bone metabolism were significantly enriched, with a number of genes shared among them (FGFR2, WNT5A, WNT3, ROR2, VEGFA, FBLN1, S1PR1, PRKCZ, TGFB3, and OSR1 for 1nM T3; and FZD1, SMAD6, NOG, NEO1, and ENG for 10 nm T3). An osteoblast-related search in the literature regarding this set of genes suggests that both T3 doses are unfavorable for osteoblast development, mainly hindering BMP and canonical and non-canonical WNT signaling. Conclusions: Therefore, this study provides new directions toward the elucidation of the mechanisms of T3 action on osteoblast metabolism, with potential future implications for the treatment of endocrine-related bone pathologies.

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Cnidarians are fascinating creatures at the base of metazoan evolution possessing an almost unlimited regeneration capacity that has attracted the interest of researchers, from Abraham Trembley's discovery of regeneration to the present. They share a simple body plan and a high morphogenetic plasticity that has led to a broad spectrum of life cycles. With molecular genomics it became unequivocally clear that Cnidaria are the sister group of the Bilateria and how similar their molecular toolkit is to that of more complex animals. This has renewed interest in these simple animals, which have had an important role in the establishment of fundamental concepts for developmental biologists from the beginning. This review focuses on our current understanding of signaling centers (organizers) and morphogenetic gradients in cnidarians and how they relate to the emergence of the bilaterian body axes. The data are largely based on the cnidarian models Hydra and Nematostella and are supported by new studies on forms with a complete cnidarian life cycle, such as the medusozoans Aurelia and Clytia. Molecular studies on cnidarian development have revealed the existence of an ancient Wnt signaling center at the site of gastrulation, which was instrumental for the formation of a primary body axis and can be traced back to the common ancestor of bilaterian and non-bilaterian animals. New molecular data also suggest that the molecular vectors for the dorso-ventral and left-right body axis in bilaterians, Bmp and Nodal signaling, respectively, were already present but had different fates in the two clades. The close link of developmental processes in bilaterians and cnidarians but also their distinct differences make cnidarians an indispensable model for tackling fundamental questions in developmental biology from patterning, regeneration and other recent molecular approaches to theoretical concepts.

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Background: Cancer is considered one of the most lethal diseases worldwide. Venous thromboembolism (VTE) is the second leading cause of death in cancer patients. As one of the most reproducible predictors of thromboembolism, the D-dimer level is commonly considered by oncologists. Previous studies have demonstrated that the most correlated genes at the D-dimer level are F3, F5 and FGA. Methods: Using data from TCGA and multiple webtools, including GEPIA2, UALCAN, TIMER2.0, Kaplan-Meier Plotter and CIBERSORTx, we analyzed the tumor mutation burden (TMB), microsatellite instability (MSI) and functions of D-dimer-related genes in cancer. Validation was conducted via quantitative real-time polymerase chain reaction (qRT-PCR) and independent GEO + GTEx cohort. All statistical analyses were performed in R software and GraphPad Prism 9. Results: F3, F5 and FGA were expressed differently in multiple cancer types. TMB, MSI and anti-PD1/PDL1 therapy responses were correlated with D-dimer-related gene expression. D-Dimer-related genes expression affect the survival of cancer patients. F3 and F5 functioned in TGF-beta signaling. F3 and F5 were related to immunity and affected the fraction of CD8+ T cells by upregulating the TGF-beta signaling pathway, forming an F3, F5/TGF-beta signaling/CD8+ T cell axis. Conclusion: F3, F5 and FGA serve as satisfactory GC multibiomarkers and potentially influence the immune microenvironment and survival of cancer patients by influencing TGF-beta signaling.

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Sex is a major biological factor in the development and physiology of a sexual reproductive organism, and its role in the growing process is needed to be investigated in various species. We compare blood transcriptome between 5 males and 5 females in 4-week-old Rhode Island Red chickens and perform functional annotation of differentially expressed genes (DEGs). The results are as follows. 141 and 109 DEGs were located in autosomes and sex chromosomes, respectively. The gene ontology (GO) terms are significantly (p < 0.05) enriched, which were limb development, inner ear development, positive regulation of dendrite development, the KEGG pathway the TGF-beta signaling pathway, and melanogenesis (p < 0.05). These pathways are related to morphological maintenance and growth of the tissues. In addition, the SMAD2W and the BMP5 were involved in the TGF-beta signaling pathway, and both play an important role in maintaining tissue development. The major DEGs related to the development of neurons and synapses include the up-regulated NRN1, GDF10, SLC1A1, BMP5, NBEA, and NRXN1. Also, 7 DEGs were validated using RT-qPCR with high correlation (r 2 = 0.74). In conclusion, the differential expression of blood tissue in the early growing chicken was enriched in TGF-beta signaling and related to the development of neurons and synapses including SMAD2W and BMP5. These results suggest that blood in the early growing stage is differentially affected in tissue development, nervous system, and pigmentation by sex. For future research, experimental characterization of DEGs and a holistic investigation of various tissues and growth stages will be required.

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BACKGROUND: Mesothelial E- and P-selectins substantially mediate the intraperitoneal spread of Pancreatic ductal adenocarcinoma (PDA) cells in xenograft models. In the absence of selectins in the host, the integrin subunit alpha-V (ITGAV, CD51) was upregulated in the remaining metastatic deposits. Here we present the first experimental study to investigate if ITGAV plays a functional role in PDA tumor growth and progression with a particular focus on intraperitoneal carcinomatosis. METHODS: Knockdown of ITGAV was generated using an RNA interference-mediated approach in two PDA cell lines. Tumor growth, intraperitoneal and distant metastasis were analyzed in a xenograft model. Cell lines were characterized in vitro. Gene expression of the xenograft tumors was analyzed. Patient samples were histologically classified and associations to survival were evaluated. RESULTS: The knockdown of ITGAV in PDA cells strongly reduces primary tumor growth, peritoneal carcinomatosis and spontaneous pulmonary metastasis. ITGAV activates latent TGF-ß and thereby drives epithelial-mesenchymal transition. Combined depletion of ITGAV on the tumor cells and E- and P-selectins in the tumor-host synergistically almost abolishes intraperitoneal spread. Accordingly, high expression of ITGAV in PDA cells was associated with reduced survival in patients. CONCLUSION: Combined depletion of ITGAV in PDA cells and E- and P-selectins in host mice massively suppresses intraperitoneal carcinomatosis of PDA cells xenografted into immunodeficient mice, confirming the hypothesis of a partly redundant adhesion cascade of metastasizing cancer cells. Our data strongly encourage developing novel therapeutic approaches for the combined targeting of E- and P-selectins and ITGAV in PDA.

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Bisphenol A (BPA) is a high-production-volume monomer for the manufacture of a wide variety of polycarbonate plastics and resins. Evidence suggests BPA can induce carcinogenesis, reproductive toxicity, abnormal inflammatory or immune response, and developmental disorders of the brain or nervous system. However, whether BPA affects the very same basic molecular processes in all the in vivo and in vitro systems employed to exert its molecular mechanisms of toxicity remains to be clarified. In this study, we collected multi-source global transcriptomics datasets for BPA-exposed organisms and cells, and evaluated the adverse effects of BPA by using data integration and gene functional enrichment analyses. We found that BPA may affect basic cellular processes, such as cell growth, survival, proliferation, differentiation, and apoptosis, independent of species and specific in vivo or in vitro systems. Mechanistically, BPA could regulate cell-extra cellular matrix interactions via challenging TGF-beta signaling pathways. Furthermore, we compared our in vitro BPA-dependent mouse embryoid body (EB) global differentiation transcriptomics with all the other datasets. We verified the EB-based toxicological system could recapitulate several in vivo and other in vitro findings very efficiently, and in a less time- and resource-consuming fashion. Taken together, this study emphasizes the utility of meta-analyses to understand common molecular mechanisms of toxicity of synthetic chemicals.

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Age-related macular degeneration (AMD) is the most common cause of central vision loss among elderly populations in industrialized countries. Genome-wide association studies have consistently associated two genomic loci with progression to late-stage AMD: the complement factor H (CFH) locus on chromosome 1q31 and the age-related maculopathy susceptibility 2-HtrA serine peptidase 1 (ARMS2-HTRA1) locus on chromosome 10q26. While the CFH risk variant has been shown to alter complement activity, the ARMS2-HTRA1 risk haplotype remains enigmatic due to high linkage disequilibrium and inconsistent functional findings spanning two genes that are plausibly causative for AMD risk. In this review, we detail the genetic and functional evidence used to support either ARMS2 or HTRA1 as the causal gene for AMD risk, emphasizing both the historical development and the current understanding of the ARMS2-HTRA1 locus in AMD pathogenesis. We conclude by summarizing the evidence in favor of HTRA1 and present our hypothesis whereby HTRA1-derived ECM fragments mediate AMD pathogenesis.

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microRNAs (miRNAs or miRs) play key roles in different stages of chronic myeloid leukemia (CML) pathogenesis. The present study aimed to demonstrate whether miR-155 enables CD34+ CML cells to escape from the growth-inhibitory effects of TGF-ß1 and bone morphogenetic protein (BMP) signaling. Among differentially expressed miRNAs in CD34+ CML cells, miR-155 was highly up-regulated. QRT-PCR revealed an inverse correlation between miR-155 and two key members of the TGF-ß pathway-TGF-ßR2 and SMAD5. Results showed that SMAD5 is not only up-regulated through BMPs treatment, but recombinant TGF-ß1 can also induce SMAD5 in CML cells. We also demonstrated that TGF-ß1-mediated phosphorylation of SMAD1/5 was abolished by pre-treatment with the blocking TGF-ßR2 antibody, suggesting a possible involvement of TGF-ßR2. Additionally, overexpression of miR-155 significantly promoted the proliferation rate of CD34+ CML cells. Results showed that siRNA-mediated knockdown of SMAD5 had a promoting effect on CD34+ CML cell proliferation, suggesting that SMAD5 knock-down recapitulates the proliferative effects of miR-155. Importantly, TGF-ß1 and BMP2/4 treatment had inhibitory effects on cell proliferation; however, miR-155 overexpression enabled CD34+ CML cells to evade the anti-proliferative effects of TGF-ß1 and BMPs. Consistently, down-regulation of miR-155 augmented the promoting effects of TGF-ß1 and BMP signaling on inducing apoptosis in CD34+ CML stem cells. Our findings demonstrated that targeting of SMAD5 and TGF-ßR2 links miR-155 to TGF-ß signaling in CML. Overexpression of miR-155 enables CD34+ CML cells to evade growth-inhibitory effects of the TGF-ß1 and BMP signaling, providing new perspectives for miR-155 as a therapeutic target for CML.

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Tumor suppressor genes represent a major class of oncogenic drivers. However, direct targeting of loss-of-function tumor suppressors remains challenging. To address this gap, we explored a variant-directed chemical biology approach to reverse the lost function of tumor suppressors using SMAD4 as an example. SMAD4, a central mediator of the TGF-ß pathway, is recurrently mutated in many tumors. Here, we report the development of a TR-FRET technology that recapitulated the dynamic differential interaction of SMAD4 and SMAD4R361H with SMAD3 and identified Ro-31-8220, a bisindolylmaleimide derivative, as a SMAD4R361H/SMAD3 interaction inducer. Ro-31-8220 reactivated the dormant SMAD4R361H-mediated transcriptional activity and restored TGF-ß-induced tumor suppression activity in SMAD4 mutant cancer cells. Thus, demonstration of Ro-31-8220 as a SMAD4R361H/SMAD3 interaction inducer illustrates a general strategy to reverse the lost function of tumor suppressors with hypomorph mutations and supports a systematic approach to develop small-molecule protein-protein interaction (PPI) molecular glues for biological insights and therapeutic discovery.

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Activin A and other TGFß family members have been shown to exhibit a certain degree of promiscuity between their family of receptors. We previously developed an efficient differentiation protocol using Activin A to obtain medium spiny neurons (MSNs) from human pluripotent stem cells (hPSCs). However, the mechanism underlying Activin A-induced MSN fate specification remains largely unknown. Here we begin to tease apart the different components of TGFß pathways involved in MSN differentiation and demonstrate that Activin A acts exclusively via ALK4/5 receptors to induce MSN progenitor fate during differentiation. Moreover, we show that Alantolactone, an indirect activator of SMAD2/3 signalling, offers an alternative approach to differentiate hPSC-derived forebrain progenitors into MSNs. Further fine tuning of TGFß pathway by inhibiting BMP signalling with LDN193189 achieves accelerated MSN fate specification. The present study therefore establishes an essential role for TGFß signalling in human MSN differentiation and provides a fully defined and highly adaptable small molecule-based protocol to obtain MSNs from hPSCs.