Paracrine TGF-β signaling counterbalances BMP-mediated repression in hair follicle stem cell activation

Hair follicle (HF) regeneration begins when communication between quiescent epithelial stem cells (SCs) and underlying mesenchymal dermal papillae (DP) generates sufficient activating cues to overcome repressive BMP signals from surrounding niche cells. Here, we uncover a hitherto unrecognized DP transmitter, TGF-β2, which activates Smad2/3 transiently in HFSCs concomitant with entry into tissue regeneration. This signaling is critical: HFSCs that cannot sense TGF-β exhibit significant delays in HF regeneration, whereas exogenous TGF-β2 stimulates HFSCs in vivo and in vitro. By engineering TGF-β- and BMP-reporter mice, we show that TGF-β2 signaling antagonizes BMP signaling in HFSCs but not through competition for limiting Smad4-coactivator. Rather, our microarray, molecular, and genetic studies unveil Tmeff1 as a direct TGF-β2/Smad2/3 target gene, expressed by activated HFSCs and physiologically relevant in restricting and lowering BMP thresholds in the niche. Connecting BMP activity to an SC's response to TGF-βs may explain why these signaling factors wield such diverse cellular effects.     

TGF-β family signaling in stem cells

Background

The diversity of cell types and tissue types that originate throughout development derives from the differentiation potential of embryonic stem cells and somatic stem cells. While the former are pluripotent, and thus can give rise to a full differentiation spectrum, the latter have limited differentiation potential but drive tissue remodeling. Additionally cancer tissues also have a small population of self-renewing cells with stem cell properties. These cancer stem cells may arise through dedifferentiation from non-stem cells in cancer tissues, illustrating their plasticity, and may greatly contribute to the resistance of cancers to chemotherapies.

Scope of review

The capacity of the different types of stem cells for self-renewal, the establishment and maintenance of their differentiation potential, and the selection of differentiation programs are greatly defined by the interplay of signaling molecules provided by both the stem cells themselves, and their microenvironment, the niche. Here we discuss common and divergent roles of TGF-β family signaling in the regulation of embryonic, reprogrammed pluripotent, somatic, and cancer stem cells.

Major conclusions

Increasing evidence highlights the similarities between responses of normal and cancer stem cells to signaling molecules, provided or activated by their microenvironment. While TGF-β family signaling regulates stemness of normal and cancer stem cells, its effects are diverse and depend on the cell types and physiological state of the cells.

General significance

Further mechanistic studies will provide a better understanding of the roles of TGF-β family signaling in the regulation of stem cells. These basic studies may lead to the development of a new therapeutic or prognostic strategies for the treatment of cancers. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Negative regulation of TGF-β signaling in development

The TGF-β superfamily members have important roles in controlling patterning and tissue formation in both invertebrates and vertebrates. Two types of signal transducers, receptors and Smads, mediate the signaling to regulate expression of their target genes. Despite of the relatively simple signal transduction pathway, many modulators have been found to contribute to a tight regulation of this pathway in a variety of mechanisms. This article reviews the negative regulation of TGF-β signaling with focus on its roles in vertebrate development.     

Disruption of TGF-β signaling in smooth muscle cell prevents flow-induced vascular remodeling

Transforming growth factor-β (TGF-β) signaling has been prominently implicated in the pathogenesis of vascular remodeling, especially the initiation and progression of flow-induced vascular remodeling. Smooth muscle cells (SMCs) are the principal resident cells in arterial wall and are critical for arterial remodeling. However, the role of TGF-β signaling in SMC for flow-induced vascular remodeling remains unknown. Therefore, the goal of our study was to determine the effect of TGF-β pathway in SMC for vascular remodeling, by using a genetical smooth muscle-specific (SM-specific) TGF-β type II receptor (Tgfbr2) deletion mice model. Mice deficient in the expression of Tgfbr2 (MyhCre.Tgfbr2^f/f) and their corresponding wild-type background mice (MyhCre.Tgfbr2^WT/WT) underwent partial ligation of left common carotid artery for 1, 2, or 4 weeks. Then the carotid arteries were harvested and indicated that the disruption of Tgfbr2 in SMC provided prominent inhibition of vascular remodeling. And the thickening of carotid media, proliferation of SMC, infiltration of macrophage, and expression of matrix metalloproteinase (MMP) were all significantly attenuated in Tgfbr2 disruption mice. Our study demonstrated, for the first time, that the TGF-β signaling in SMC plays an essential role in flow-induced vascular remodeling and disruption can prevent this process.     

TGF-β mediates proinflammatory seminal fluid signaling in human cervical epithelial cells

The cervix is central to the female genital tract immune response to pathogens and foreign male Ags introduced at coitus. Seminal fluid profoundly influences cervical immune function, inducing proinflammatory cytokine synthesis and leukocyte recruitment. In this study, human Ect1 cervical epithelial cells and primary cervical cells were used to investigate agents in human seminal plasma that induce a proinflammatory response. TGF-β1, TGF-β2, and TGF-β3 are abundant in seminal plasma, and Affymetrix microarray revealed that TGF-β3 elicits changes in Ect1 cell expression of several proinflammatory cytokine and chemokine genes, replicating principal aspects of the Ect1 response to seminal plasma. The differentially expressed genes included several induced in the physiological response of the cervix to seminal fluid in vivo. Notably, all three TGF-β isoforms showed comparable ability to induce Ect1 cell expression of mRNA and protein for GM-CSF and IL-6, and TGF-β induced a similar IL-6 and GM-CSF response in primary cervical epithelial cells. TGF-β neutralizing Abs, receptor antagonists, and signaling inhibitors ablated seminal plasma induction of GM-CSF and IL-6, but did not alter IL-8, CCL2 (MCP-1), CCL20 (MIP-3α), or IL-1α production. Several other cytokines present in seminal plasma did not elicit Ect1 cell responses. These data identify all three TGF-β isoforms as key agents in seminal plasma that signal induction of proinflammatory cytokine synthesis in cervical cells. Our findings suggest that TGF-β in the male partner's seminal fluid may influence cervical immune function after coitus in women, and potentially be a determinant of fertility, as well as defense from infection.     

The notch and TGF-β signaling pathways contribute to the aggressiveness of clear cell renal cell carcinoma

Background

Despite recent progress, therapy for metastatic clear cell renal cell carcinoma (CCRCC) is still inadequate. Dysregulated Notch signaling in CCRCC contributes to tumor growth, but the full spectrum of downstream processes regulated by Notch in this tumor form is unknown.

Methodology/Principal Findings

We show that inhibition of endogenous Notch signaling modulates TGF-β dependent gene regulation in CCRCC cells. Analysis of gene expression data representing 176 CCRCCs showed that elevated TGF-β pathway activity correlated significantly with shortened disease specific survival (log-rank test, p = 0.006) and patients with metastatic disease showed a significantly elevated TGF-β signaling activity (two-sided Student''s t-test, p = 0.044). Inhibition of Notch signaling led to attenuation of both basal and TGF-β1 induced TGF-β signaling in CCRCC cells, including an extensive set of genes known to be involved in migration and invasion. Functional analyses revealed that Notch inhibition decreased the migratory and invasive capacity of CCRCC cells.

Conclusion

An extensive cross-talk between the Notch and TGF-β signaling cascades is present in CCRCC and the functional properties of these two pathways are associated with the aggressiveness of this disease.     

Disruption of TGF-β signaling in smooth muscle cell prevents elastase-induced abdominal aortic aneurysm

Transforming growth factor-β (TGF-β) signaling has been significantly implicated in the pathogenesis of aneurysm, prominently the initiation and progression of abdominal aortic aneurysm (AAA). Vascular smooth muscle cell (SMC) is the principal resident cell in aortic wall and is essential for its structure and function. However, the role of TGF-β pathway in SMC for the formation of AAA remains unknown. Therefore, the goal of the present study was to investigate the effect of TGF-β pathway in SMC for AAA pathogenesis, by using a genetical smooth muscle-specific (SM-specific) TGF-βtype II receptor (Tgfbr2) disruption animal model. Mice deficient in the expression of Tgfbr2 (MyhCre.Tgfbr2^f/f and MyhCre.Tgfbr2^WT/f) and their corresponding wild-type background mice (MyhCre.Tgfbr2^WT/WT) underwent AAA induction by infrarenal peri-adventitial application of elastase. Fourteen days after elastase treatment, the aortas were analyzed and indicated that disruption of 1 or 2 alleles of Tgfbr2 in SMC provided markedly step-wise protection from AAA formation. And elastin degradation, medial SMC loss, macrophage infiltration, and matrix metalloproteinases (MMP) expression were all significantly reduced in Tgfbr2 deletion mice. Our study demonstrated, for the first time, that the TGF-β signaling pathway in SMC plays a critical role in AAA and disruption can prevent the aneurysm formation.     

Nuclear translocation of Skp2 facilitates its destruction in response to TGFβ signaling

Skp2, a F-box protein that determines the substrate specificity for SCF ubiquitin ligase, has recently been demonstrated to be degraded by Cdh1/APC in response to TGFβ signaling. The TGFβ-induced Skp2 proteolysis results in the stabilization of p27 that is necessary to facilitate TGFβ cytostatic effect. Previous observation from immunocytochemistry indicates that Cdh1 principally localizes in the nucleus while Skp2 mainly localizes in the cytosol, which leaves us a puzzle on how Skp2 is recognized and then ubiquitylated by Cdh1/APC in response to TGFβ stimulation. Here, we report that Skp2 is rapidly translocated from the cytosol to the nucleus upon the cellular stimulation with TGFβ. Using a combinatorial approach of immunocytochemistry, biochemical-fraction-coupled immunoprecipitation, mutagenesis as well as protein degradation assay, we have demonstrated that the TGFβ-induced Skp2 nucleus translocation is critical for TGFβ cytostatic effect that allows physical interaction between Cdh1 and Skp2 and in turn facilitates the Skp2 ubquitylation by Cdh1/APC. Disruption of nuclear localization motifs on Skp2 stabilizes Skp2 in the presence of TGFβ signaling, which attenuates TGFβ-induced p27 accumulation and antagonizes TGFβ-induced growth inhibition. Our finding reveals a cellular mechanism that facilitates Skp2 ubiquitylation by Cdh1/APC in response to TGFβ.Key words: Skp2, nuclear translocation, ubiquitylation, TGFβ     

Inhibiting TGF-β signaling in hepatocellular carcinoma

One of the main complications in patients with liver fibrosis is the development of hepatocellular carcinoma (HCC). An understanding of the molecular mechanisms leading to HCC is important in order to be able to design new pharmacological agents serving either to prevent or mitigate the outcome of this malignancy. The transforming growth factor-beta (TGF-β) cytokine and its isoforms initiate a signaling cascade which is closely linked to liver fibrosis, cirrhosis and subsequent progression to HCC. Because of its role in these stages of disease progression, TGF-β appears to play a unique role in the molecular pathogenesis of HCC. Thus, it is a promising target for pharmacological treatment strategies. Recent studies have shown that inhibition of TGF-β signaling results in multiple synergistic down-stream effects which will likely improve the clinical outcome in HCC. We also review a number of TGF-β inhibitors, most of which are still in a preclinical stage of development, but may soon be available for trial in HCC patients. Hence, it is anticipated that there will soon be new agents available for clinical investigations to evaluate the role of the TGF-β-associated signaling in this deadly cancer.     

Intercellular variation in signaling through the TGF-β pathway and its relation to cell density and cell cycle phase

Fundamental open questions in signal transduction remain concerning the sequence and distribution of molecular signaling events among individual cells. In this work, we have characterized the intercellular variability of transforming growth factor β-induced Smad interactions, providing essential information about TGF-β signaling and its dependence on the density of cell populations and the cell cycle phase. By employing the recently developed in situ proximity ligation assay, we investigated the dynamics of interactions and modifications of Smad proteins and their partners under native and physiological conditions. We analyzed the kinetics of assembly of Smad complexes and the influence of cellular environment and relation to mitosis. We report rapid kinetics of formation of Smad complexes, including native Smad2-Smad3-Smad4 trimeric complexes, in a manner influenced by the rate of proteasomal degradation of these proteins, and we found a striking cell to cell variation of signaling complexes. The single-cell analysis of TGF-β signaling in genetically unmodified cells revealed previously unknown aspects of regulation of this pathway, and it provided a basis for analysis of these signaling events to diagnose pathological perturbations in patient samples and to evaluate their susceptibility to drug treatment.     

Differential expression of secreted phosphoprotein 1 in response to estradiol-17β and in ovarian tumors in chickens

Roundabout 2 (Robo2) is a member of the membrane protein receptor family. The chemorepulsive effect of Slit2-Robo2 signaling plays vital roles in nervous system development and neuron migration. Slit2-Robo2 signaling is also important for maintaining the normal morphogenesis of the kidney and urinary collecting system, especially for the branching of the ureteric bud (UB) at the proper site. Slit2 or Robo2 mouse mutants exhibit multilobular kidneys, multiple ureters, and dilatation of the ureter, renal pelvis, and collecting duct system, which lead to vesicoureteral reflux. To understand the effect of Robo2 on kidney development, we used microinjection and electroporation to overexpress GFP-Robo2 in an in vitro embryonic kidney model. Our results show reduced UB branching and decreased glomerular number after in vitro Robo2 overexpression in the embryonic kidneys. We found fewer metanephric mesenchymal (MM) cells surrounding the UB but no abnormal morphology in the branching epithelial UB. Meanwhile, no significant change in MM proliferation or apoptosis was observed. These findings indicate that Robo2 is involved in the development of embryonic kidneys and that the normal expression of Robo2 can help maintain proper UB branching and glomerular morphogenesis. Overexpression of Robo2 leads to reduced UB branching caused by fewer surrounding MM cells, but MM cell apoptosis is not involved in this effect. Our study demonstrates that overexpression of Robo2 by microinjection in embryonic kidneys is an effective approach to study the function of Robo2.     

TGF-β regulates β-catenin signaling and osteoblast differentiation in human mesenchymal stem cells

Human adult bone marrow-derived skeletal stem cells a.k.a mesenchymal stem cells (hMSCs) have been shown to be precursors of several different cellular lineages, including osteoblast, chondrocyte, myoblast, adipocyte, and fibroblast. Several studies have shown that cooperation between transforming growth factor β (TGF-β) and Wnt/β-catenin signaling pathways plays a role in controlling certain developmental events and diseases. Our previous data showed that agents like TGF-β, cooperation with Wnt signaling, promote chondrocyte differentiation at the expense of adipocyte differentiation in hMSCs. In this study, we tested mechanisms by which TGF-β activation of β-catenin signaling pathway and whether these pathways interact during osteoblast differentiation of hMSCs. With selective small chemical kinase inhibitors, we demonstrated that TGF-β1 requires TGF-β type I receptor ALK-5, Smad3, phosphoinositide 3-kinases (PI3K), and protein kinase A (PKA) to stabilize β-catenin, and needs ALK-5, PKA, and JNK to inhibit osteoblastogenesis in hMSCs. Knockdown of β-catenin with siRNA stimulated alkaline phosphatase activity and antagonized the inhibitory effects of TGF-β1 on bone sialoprotein (BSP) expression, suggested that TGF-β1 cooperated with β-catenin signaling in inhibitory of osteoblastogenesis in hMSCs. In summary, TGF-β1 activates β-catenin signaling pathway via ALK-5, Smad3, PKA, and PI3K pathways, and modulates osteoblastogenesis via ALK5, PKA, and JNK pathways in hMSCs; the interaction between TGF-β and β-catenin signaling supports the view that β-catenin signaling is a mediator of TGF-β's effects on osteoblast differentiation of hMSCs.