Influence Of Activin Research Articles

P157 Differentiation of Tonsil-derived mesenchymal stem cells to intestinal stem cells-like cells for cell therapy of inflammatory bowel disease

Abstract Background Tonsil-derived mesenchymal stem cells (TMSCs) were found to exhibit a faster growth rate compared to mesenchymal stem cells derived from other tissues. This is attributed to the relatively younger age of the tonsil tissue donors. Moreover, it has been verified that TMSCs possess the capability to undergo differentiation into endoderm lineages, giving rise to cell types like parathyroid glands and insulin-producing cells. In this study, we explored the possibility of differentiation of TMSCs into intestinal stem cells-like cells (ISC-like cells), a cell type of endoderm lineages for utilizing these cells for therapeutic applications in inflammatory bowel disease (IBD). Methods TMSCs were cultivated in DMEM (10% FBS) and used in the experiment when 90-100% confluency was observed. Adipose-derived mesenchymal stem cells (AMSCs) were used as the control for assessing the differentiation efficacy of TMSCs. The morphology of cells was verified at each differentiation stage, and the expression of endoderm and intestinal stem cell markers was confirmed using real-time PCR (qPCR), western blotting, and immunofluorescence (IF) staining. Results Subjected to 100ng/ml of Activin A for endoderm differentiation and cultured for 2 to 5 days, TMSCs exhibited an upregulation in endoderm markers (SOX17, FOXA2), as confirmed by real-time PCR. Specifically, under the influence of Activin A at 100ng/ml and cultured for 5 days, there was a significant increase in the expression of SOX17 (pre-differentiation vs. endoderm-differentiation, 0.999 vs. 3.610, P=0.0394) and FOXA2 (1.047 vs. 3.912, P=0.0297). Following the endoderm differentiation phase, the medium was changed to include 500ng/ml of FGF4 and 500ng/ml of Wnt3a for a 7-day period. Post-culture, the expression level of Lgr5, an intestinal stem cell (ISC) marker, was significantly increased in TMSCs (1.061 vs. 134.0, P<0.001). Notably, AMSCs displayed a lower expression rate compared to TMSCs (TMSCs vs. AMSCs, 134.0 vs. 9.819, P<0.001). Moreover, the spheroids formation was observed 7 days after the initiation of FGF4 and Wnt3a treatment, a phenomenon absent in AMSCs differentiation. Immunofluorescence staining for differentiation markers further validated these findings: the expression of SOX17, an endoderm marker, and the fluorescence density of LGR5, an ISC marker, were notably higher in TMSCs compared to AMSCs. Protein expression levels also confirmed the upregulation of each marker in TMSCs. Conclusion This study validated the potential of TMSCs to differentiate into ISC-like cells, with a higher efficiency observed compared to AMSCs. The prospect of differentiating into ISC positions TMSCs as promising candidates for use as a tool in IBD.

Hepatic Activin E mediates liver-adipose inter-organ communication, suppressing adipose lipolysis in response to elevated serum fatty acids

ObjectiveThe liver is a central regulator of energy metabolism exerting its influence both through intrinsic processing of substrates such as glucose and fatty acid as well as by secreting endocrine factors, known as hepatokines, which influence metabolism in peripheral tissues. Human genome wide association studies indicate that a predicted loss-of-function variant in the Inhibin βE gene (INHBE), encoding the putative hepatokine Activin E, is associated with reduced abdominal fat mass and cardiometabolic disease risk. However, the regulation of hepatic Activin E and the influence of Activin E on adiposity and metabolic disease are not well understood. Here, we examine the relationship between hepatic Activin E and adipose metabolism, testing the hypothesis that Activin E functions as part of a liver-adipose, inter-organ feedback loop to suppress adipose tissue lipolysis in response to elevated serum fatty acids and hepatic fatty acid exposure. MethodsThe relationship between hepatic Activin E and non-esterified fatty acids (NEFA) released from adipose lipolysis was assessed in vivo using fasted CL 316,243 treated mice and in vitro using Huh7 hepatocytes treated with fatty acids. The influence of Activin E on adipose lipolysis was examined using a combination of Inhbe knockout mice, a mouse model of hepatocyte-specific overexpression of Activin E, and mouse brown adipocytes treated with Activin E enriched media. ResultsIncreasing hepatocyte NEFA exposure in vivo by inducing adipose lipolysis through fasting or CL 316,243 treatment increased hepatic Inhbe expression. Similarly, incubation of Huh7 human hepatocytes with fatty acids increased expression of INHBE. Genetic ablation of Inhbe in mice increased fasting circulating NEFA and hepatic triglyceride accumulation. Treatment of mouse brown adipocytes with Activin E conditioned media and overexpression of Activin E in mice suppressed adipose lipolysis and reduced serum FFA levels, respectively. The suppressive effects of Activin E on lipolysis were lost in CRISPR-mediated ALK7 deficient cells and ALK7 kinase deficient mice. Disruption of the Activin E-ALK7 signaling axis in Inhbe KO mice reduced adiposity upon HFD feeding, but caused hepatic steatosis and insulin resistance. ConclusionsTaken together, our data suggest that Activin E functions as part of a liver-adipose feedback loop, such that in response to increased serum free fatty acids and elevated hepatic triglyceride, Activin E is released from hepatocytes and signals in adipose through ALK7 to suppress lipolysis, thereby reducing free fatty acid efflux to the liver and preventing excessive hepatic lipid accumulation. We find that disrupting this Activin E-ALK7 inter-organ communication network by ablation of Inhbe in mice increases lipolysis and reduces adiposity, but results in elevated hepatic triglyceride and impaired insulin sensitivity. These results highlight the liver-adipose, Activin E-ALK7 signaling axis as a critical regulator of metabolic homeostasis.

The Influence of Activin on the Tumor Microenvironment in Colon Cancer Metastasis

Background and AimsTGFβ and activin A are key molecules regulating tumor growth, inflammatory responses in the TME, and have differential effects which are receptor and cell specific. Identification of the source of activin A/TGFβ production and site of signaling will be critical for directing therapeutic development. Here, we employ both mouse models and human tissue samples to 1) identify pathways in which SMAD4 mutations exacerbate tumor growth and metastasis, 2) recognize how TGFβ and activin A signaling are influencing immune responses in the TME, and 3) to use the cellular source and signaling of activin A, rather than the amount, to predict patient outcomes in CRC. We have previously shown elevated levels of circulating activin A in stage IV CRC patients and that activin signaling promotes epithelial to mesenchymal transition and migration. We now hypothesize that activin A signaling pathways have distinct effects on immune cells and tumor cells which regulate tumor development and metastasis of CRC.MethodsKnockdown of activin A was assessed in a surgical colon cancer liver metastases mouse model. The influence of the SMAD4‐dependent, canonical pathway was elucidated in a Ts4‐Cre; cApcflox;Smad4flox (TcAS) mouse model for colon cancer. Western blotting and migration assays were performed on human colon cancer cells with mutations in receptors for TGFꞵ and/or activin to determine functional interactions of the downstream pathways. We used a TMA of 1054 CRC patients with stage II and III colon cancer from the QUASAR2 cohort to correlate activin and TGFb expression with outcome.ResultsThe non‐canonical pathway pAKT is activated in ACVR2 restored HCT116+chr2 colon cancer cells, but not in ACVR2 mutated cells. Ablation of the the activin A canonical pathway target SMAD4 in mice leads to decreased survival in colon cancer mouse model and pERK is increased in SMAD4‐/‐ knock out mice and correlates with dysplasia grade. Mice harboring colonic lesions with high stroma content were positive for activin A and CAF marker α‐SMA and have shorter median survival. Activin knockdown in mouse metastasis model leads to decreased tumor growth. Patient data revealed that a high ratio of activin A relative to TGFꞵ in the tumor epithelium, but not in the stroma, is associated with an increased survival rate. Ongoing studies are being conducted to determine if activin A is being localized to immune cells interacting with the tumor epithelium which may be promoting anti‐tumor activity in these patients.ConclusionRecent clinical trials explored the feasibility of activin A and TGFβ blockade in solid tumors. Data presented here supports complex and intertwined roles for activin and TGFβ signaling in colorectal cancer, both in immune activation as well as metastatic spread of advanced cancers.

Abstract 1438: Opposing influence of activin and TGFβ on PI3K and MEK/ERK signaling in colon cancer

Abstract Introduction: Colorectal cancer (CRC) is the second deadliest cancer in the US. The overall number of new cases and deaths has decreased, most likely due to increased screening. However, once CRC has metastasized, survival rate drops dramatically. In early stage CRC, the Transforming Growth Factor (TGF) β super family is growth suppressive; in advanced disease, high TGFβ serum levels are associated with poor prognosis. Activin and TGFβ regulate cell growth, apoptosis, and cell migration involving a poorly understood switch from growth suppressive to proinvasive actions. Understanding the switch to metastatic behavior and developing therapeutic strategies and biomarkers to target this process are clinical challenges. We recently observed opposing effects of TGFβ and activin on a downstream target, the cell cycle inhibitor p21. TGFβ induces SMAD4-dependent upregulation of p21, while activin downregulation of p21 is SMAD4-independent. Here, we dissect the unique contributions of mitogenic pathways to a TGFβ or activin-induced metastatic phenotype in colon cancer. Methods: Colon cancer cell lines (+/- SMAD4) were assessed for ligand-induced PI3K and MEK/ERK pathway activation. Protein/phospho-isoform expression and association were determined following knockdown and pharmacologic inhibition of pathway members. TGFβ and activin influence on cell migration via mitogenic pathways was assessed through use of small molecule inhibitors of signaling members. Epithelial mesenchymal transition (EMT) was assessed by altered expression of epithelial and mesenchymal markers. Pathway activation was compared in intestinal tumors from AOM/DSS treated ACVR2 knockout and ACVR2 wild type mice. Mitogenic signaling/growth factor status and p21 localization were correlated in primary colon cancers. Results: Activin, but not TGFβ, led to PI3K activation via interaction of ACVR1 and p85 independent of SMAD4, resulting in p21 downregulation. In contrast, TGFβ increased significantly p21 via MEK/ERK/SMAD4 synergy through a SMAD4-dependent mechanism. While activin induced EMT via PI3K, TGFβ induced EMT via MEK/ERK activation. In vivo, loss of ACVR2 resulted in loss of pAkt, consistent with activin-dependent PI3Ksignaling. In primary colon cancers, loss of nuclear p21 correlated with upstream activation of activin/PI3K while nuclear p21 was associated with TGFβ/MEK/ERK pathway activation. Conclusions: Activin and TGFβ diverge in their pro-migratory, SMAD4-independent signaling in colon cancer and utilize distinct mitogenic signaling to affect p21 localization. p21 localization in colon cancer may warrant further validation as a therapeutic biomarker for targeting TGFβ family receptors. Citation Format: Jessica I. Bauer, Naomi Akagi, Ozkan Ozden, Daniel R. Principe, Timothy Carroll, Seung Hyun Baik, Martina E. Spehlmann, Lars Eckmann, Paul J. Grippo, Barbara Jung. Opposing influence of activin and TGFβ on PI3K and MEK/ERK signaling in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1438. doi:10.1158/1538-7445.AM2015-1438

Influence of Activin A Supplementation During Human Embryonic Stem Cell Derivation on Germ Cell Differentiation Potential

Human embryonic stem cells (hESCs) are more similar to "primed" mouse epiblast stem cells (mEpiSCs). mEpiSCs, which are derived in Activin A, show an increased propensity to form primordial germ cell (PGC)-like cells in response to bone morphogenic protein 4 (BMP4). Hence, we hypothesized that hESCs derived in the presence of Activin A may be more competent in differentiating towards PGC-like cells after supplementation with BMP4 compared to standard hESC lines. We were able to successfully derive two hESC lines in the presence of Activin A, which were pluripotent and showed higher base levels of STELLA and cKIT compared to standard hESC lines derived without Activin A addition. Furthermore, upon differentiation as embryoid bodies in the presence of BMP4, we observed upregulation of VASA at day 7, both at the transcript and protein level compared to standard hESC lines, which appeared to take longer time for PGC specification. Unlike other hESC lines, nuclear pSMAD2/3 presence confirmed that Activin signalling was switched on in Activin A-derived hESC lines. They were also responsive to BMP4 based on nuclear detection of pSMAD1/5/8 and showed endodermal differentiation as a result of GATA-6 expression. Hence, our results provide novel insights into the impact of hESC derivation in the presence of Activin A and its subsequent influence on germ cell differentiation potential in vitro.

Activin A and retinoic acid synergize in cyclooxygenase-1 and thromboxane synthase induction during differentiation of J774.1 macrophages.

The murine macrophage cell line J774.1 was used to study the development of prostanoid biosynthesis under the influence of activin A and retinoic acid. Treatment of cells with 3 nM activin A for 48 h increased the biosynthesis of the prostaglandins E2, D2, F2 alpha and thromboxane A2 more than fourfold due to an induction of cyclooxygenase-1 while cyclooxygenase-2 was unaffected. Transforming growth factor-beta acted in a similar way. Retinoic acid, when present alone, was without effect on the total cyclooxygenase products and only slightly changed the pattern of prostanoids. However, when coincubated with activin A, retinoic acid specifically induced the synthesis of thromboxane-A-synthase-specific mRNA and induced an increase in enzyme activity with a synergistic effect on cyclooxygenase-1 protein and mRNA. JunB, but not c-jun, mRNA expression was found under these conditions in addition to a transient c-fos mRNA increase. The combination of activin A and retinoid acid may be regarded as a differentiation model to study the development of cell-specific prostanoid patterns in macrophages and possibly other differentiating cells.

Activin modulates the intracellular signaling system activated by gonadotropin-releasing hormone: dual effect on calcium messenger system and protein kinase-C pathway

This study was performed to assess the effects of activin on intracellular mechanisms involved in GnRH action. When rat pituitary cell cultures were pretreated with activin-A (5-80 ng/ml) for 3 days, subsequent FSH and LH release (percentage of total cellular FSH and LH released during 4 h) in response to GnRH (10(-10)-10(-6) M) was not significantly different from that in cells pretreated with medium alone. In contrast, activin pretreatment increased the potency of both A23187 (Ca2+ ionophore) and phorbol 12-myristate 13-acetate [a protein kinase-C (PKC) activator] as secretagogues for FSH and LH release. FSH or LH release in response to another Ca(2+)-mobilizing secretagogue, maitotoxin (an activator of the GnRH receptor-associated Ca2+ channel), was not increased by activin. Although PKC is capable of influencing the actions of Ca2+, which is believed to be the second messenger for GnRH action, neither GnRH- nor maitotoxin-stimulated gonadotropin release was increased by activin even when the influence of activin on PKC was eliminated by the addition of a PKC inhibitor (staurosporine; 100 nM) during the final 30 min of the 3-day pretreatment period. These results indicate that although activin does not influence GnRH action with regard to gonadotropin release, it increases the sensitivity of the system regulating gonadotropin release to increases in cytosolic Ca2+ concentrations and PKC activation. Furthermore, activin appears to exhibit an inhibitory effect(s) at some point(s) in GnRH action in a PKC-independent manner, which could be responsible for opposing the increased sensitivity of the gonadotrope to Ca2+. The differential effects of activin on gonadotropin release in response to Ca(2+)-mobilizing secretagogues (ionophore and maitotoxin) raise the possibility that the activity of the GnRH receptor-associated Ca2+ channel may be suppressed by activin.

Activin modulates the intracellular signaling system activated by gonadotropin-releasing hormone: dual effect on calcium messenger system and protein kinase-C pathway.

This study was performed to assess the effects of activin on intracellular mechanisms involved in GnRH action. When rat pituitary cell cultures were pretreated with activin-A (5-80 ng/ml) for 3 days, subsequent FSH and LH release (percentage of total cellular FSH and LH released during 4 h) in response to GnRH (10(-10)-10(-6) M) was not significantly different from that in cells pretreated with medium alone. In contrast, activin pretreatment increased the potency of both A23187 (Ca2+ ionophore) and phorbol 12-myristate 13-acetate [a protein kinase-C (PKC) activator] as secretagogues for FSH and LH release. FSH or LH release in response to another Ca(2+)-mobilizing secretagogue, maitotoxin (an activator of the GnRH receptor-associated Ca2+ channel), was not increased by activin. Although PKC is capable of influencing the actions of Ca2+, which is believed to be the second messenger for GnRH action, neither GnRH- nor maitotoxin-stimulated gonadotropin release was increased by activin even when the influence of activin on PKC was eliminated by the addition of a PKC inhibitor (staurosporine; 100 nM) during the final 30 min of the 3-day pretreatment period. These results indicate that although activin does not influence GnRH action with regard to gonadotropin release, it increases the sensitivity of the system regulating gonadotropin release to increases in cytosolic Ca2+ concentrations and PKC activation. Furthermore, activin appears to exhibit an inhibitory effect(s) at some point(s) in GnRH action in a PKC-independent manner, which could be responsible for opposing the increased sensitivity of the gonadotrope to Ca2+. The differential effects of activin on gonadotropin release in response to Ca(2+)-mobilizing secretagogues (ionophore and maitotoxin) raise the possibility that the activity of the GnRH receptor-associated Ca2+ channel may be suppressed by activin.