Smad4 controls proliferation of interstitial cells in the neonatal kidney

Expansion of interstitial cells in the adult kidney is a hallmark of chronic disease, whereas their proliferation during fetal development is necessary for organ formation. An intriguing difference between adult and neonatal kidneys is that the neonatal kidney has the capacity to control interstitial cell proliferation when the target number has been reached. In this study, we define the consequences of inactivating the TGFβ/Smad response in the interstitial cell lineage. We find that pathway inactivation through loss of Smad4 leads to over-proliferation of interstitial cells regionally in the kidney medulla. Analysis of markers for BMP and TGFβ pathway activation reveals that loss of Smad4 primarily reduces TGFβ signaling in the interstitium. While TGFβ signaling is reduced in these cells, marker analysis shows that Wnt/β-catenin signaling is increased. Our analysis supports a model in which Wnt/β-catenin mediated proliferation is attenuated by TGFβ/Smad to ensure that proliferation ceases when the target number of interstitial cells has been reached in the neonatal medulla.

The mechanisms of cell invasion in cnidarian-dinoflagellate symbiosis: Learning from parasitic strategies

<p>Cnidarians such as corals, anemones and hydroids commonly form an intracellular symbiosis with photosynthetic dinoflagellates of the genus Symbiodinium. Dinoflagellate symbionts are most often obtained anew from the environment during larval development, and, once acquired reside inside host-derived vacuoles within the cnidarian gastrodermal cells. In order to gain entry to host cells, the symbionts likely interact with innate immune receptors in the extracellular matrix, the first line of defense against microbial attack. While several innate immune pathways have been described in cnidarians, little is known about the specific receptor-ligand interactions that allow the symbiont to gain entry to host cells. Furthermore, it is unclear how these pathways are involved in enabling friendly microbes to reside within host cells while maintaining an immune response to harmful pathogens. The invasion strategies of vertebrate intracellular parasites are well studied, especially those used by members of the Apicomplexa. Apicomplexan parasites have evolved mechanisms to evade immune receptors in the extracellular matrix and exploit specific receptors to their own benefit, to gain entry to host cells. Apicomplexans are closely related to dinoflagellates, both belonging to the infrakingdom Alveolata. The malaria parasite Plasmodium spp. has evolved the thromobospondin-related anonymous protein, or TRAP, that uses a thrombospondin structural homology repeat (TSR) domain to bind to a scavenger receptor (SRB1) on the hepatocyte cell surface and gain entry to the cell. This is of particular interest, as class B scavenger receptors are upregulated in the symbiotic state of two anemone species. The aims of the research presented in this thesis were to: (1) characterize the scavenger receptor (SR) repertoire in cnidarians; (2) characterize the TSR-domain-containing protein repertoire of cnidarians and their symbiotic dinoflagellates; and (3) establish, through experimental manipulation, the potential role for SR-TSR domain interactions at the onset of symbiosis in the sea anemone Aiptasia sp, a model system for the study of the cnidariandinoflagellate symbiosis. In Chapter 2, I characterized the large and diverse SR repertoire of six cnidarian species. Cnidarians lack the classic SR type-A collagen domain-containing proteins that are common in humans, however the cnidarian SR cysteine-rich domain-containing protein repertoire is expanded and diverse. Phylogenetic analysis of SR type-B proteins defines two or three distinct groups. Functional experimental data presented here show that blocking SR binding sites with fucoidan significantly reduces dinoflagellate uptake by the anemone Aiptasia sp. These data provide further evidence that SRs are important to symbiont recognition and uptake, and may be an essential component of symbiont acquisition. In Chapter 3, I investigated a SR ligand, the thrombospondin structural homology repeat, or TSR domain. In particular, I characterized the TSR-domain-containing protein repertoire of six cnidarian species and compared these proteins to vertebrate TSR proteins of known function. Searches revealed a large repertoire of TSR-domaincontaining proteins. Of particular interest is the large number of Adams metalloprotease-like proteins, a group that is common in both humans and cnidarians, suggesting that this is an ancestral TSR protein group. Phylogenetic analysis of TSR domains shows that binding motifs and 3-D folding sites are highly conserved. These data suggest that TSR domains are ancient and have changed very little in amino acid sequence from lower metazoans to vertebrates. In Chapter 4, I explored the role of TSR-domain-containing proteins at the onset of symbiosis in the model Aiptasia sp. system. In functional experiments, aposymbiotic anemones were challenged with proteins and antibodies to either block or stimulate TSR domain binding. Symbiont uptake was measured over several time-points to determine the effects on symbiont acquisition. Adding an excess of TSR-domaincontaining protein or TSR synthetic peptide increased symbiont uptake, while blocking TSR domains prevented symbiont uptake. Finally, the addition of exogenous TGFβ to TSR antibody-challenged anemones, reversed the blocking effect. These data suggest that the immune-suppressive TGFβ pathway is involved in early onset of the symbiosis. Since the TSR domain is implicated in the TGFβ pathway, these results support previous findings of the involvement of TGFβ in promoting tolerance of symbionts within the host. Apicomplexan parasites exploit scavenger receptor-TSR domain-binding to gain entry, and also use immune modulation to persist inside host cells. Data presented here suggest that dinoflagellates are utilizing the same mechanisms to form a mutualistic relationship with the cnidarian host. Overall, the work presented here provides new information about several cnidarian extracellular matrix proteins, with searches revealing large repertoires of both scavenger receptors and TSR-domain-containing proteins. Functional data suggest that both protein families are involved in the cnidarian-dinoflagellate symbiosis. Searches of the dinoflagellate genome did not find a clear dinoflagellate homologue to the apicomplexan TRAP proteins. However, this research provides further evidence that similar receptor-ligand interactions are involved in the entry of both beneficial and pathogenic microbes to host cells. These results add to growing knowledge about the complex molecular pathways that enable and support cnidarian-dinoflagellate symbiosis. An understanding of the mechanisms that support healthy symbiosis is essential when trying to predict the vitality and productivity of reef ecosystems in the face of climate change.</p>

The mechanisms of cell invasion in cnidarian-dinoflagellate symbiosis: Learning from parasitic strategies

<p>Cnidarians such as corals, anemones and hydroids commonly form an intracellular symbiosis with photosynthetic dinoflagellates of the genus Symbiodinium. Dinoflagellate symbionts are most often obtained anew from the environment during larval development, and, once acquired reside inside host-derived vacuoles within the cnidarian gastrodermal cells. In order to gain entry to host cells, the symbionts likely interact with innate immune receptors in the extracellular matrix, the first line of defense against microbial attack. While several innate immune pathways have been described in cnidarians, little is known about the specific receptor-ligand interactions that allow the symbiont to gain entry to host cells. Furthermore, it is unclear how these pathways are involved in enabling friendly microbes to reside within host cells while maintaining an immune response to harmful pathogens. The invasion strategies of vertebrate intracellular parasites are well studied, especially those used by members of the Apicomplexa. Apicomplexan parasites have evolved mechanisms to evade immune receptors in the extracellular matrix and exploit specific receptors to their own benefit, to gain entry to host cells. Apicomplexans are closely related to dinoflagellates, both belonging to the infrakingdom Alveolata. The malaria parasite Plasmodium spp. has evolved the thromobospondin-related anonymous protein, or TRAP, that uses a thrombospondin structural homology repeat (TSR) domain to bind to a scavenger receptor (SRB1) on the hepatocyte cell surface and gain entry to the cell. This is of particular interest, as class B scavenger receptors are upregulated in the symbiotic state of two anemone species. The aims of the research presented in this thesis were to: (1) characterize the scavenger receptor (SR) repertoire in cnidarians; (2) characterize the TSR-domain-containing protein repertoire of cnidarians and their symbiotic dinoflagellates; and (3) establish, through experimental manipulation, the potential role for SR-TSR domain interactions at the onset of symbiosis in the sea anemone Aiptasia sp, a model system for the study of the cnidariandinoflagellate symbiosis. In Chapter 2, I characterized the large and diverse SR repertoire of six cnidarian species. Cnidarians lack the classic SR type-A collagen domain-containing proteins that are common in humans, however the cnidarian SR cysteine-rich domain-containing protein repertoire is expanded and diverse. Phylogenetic analysis of SR type-B proteins defines two or three distinct groups. Functional experimental data presented here show that blocking SR binding sites with fucoidan significantly reduces dinoflagellate uptake by the anemone Aiptasia sp. These data provide further evidence that SRs are important to symbiont recognition and uptake, and may be an essential component of symbiont acquisition. In Chapter 3, I investigated a SR ligand, the thrombospondin structural homology repeat, or TSR domain. In particular, I characterized the TSR-domain-containing protein repertoire of six cnidarian species and compared these proteins to vertebrate TSR proteins of known function. Searches revealed a large repertoire of TSR-domaincontaining proteins. Of particular interest is the large number of Adams metalloprotease-like proteins, a group that is common in both humans and cnidarians, suggesting that this is an ancestral TSR protein group. Phylogenetic analysis of TSR domains shows that binding motifs and 3-D folding sites are highly conserved. These data suggest that TSR domains are ancient and have changed very little in amino acid sequence from lower metazoans to vertebrates. In Chapter 4, I explored the role of TSR-domain-containing proteins at the onset of symbiosis in the model Aiptasia sp. system. In functional experiments, aposymbiotic anemones were challenged with proteins and antibodies to either block or stimulate TSR domain binding. Symbiont uptake was measured over several time-points to determine the effects on symbiont acquisition. Adding an excess of TSR-domaincontaining protein or TSR synthetic peptide increased symbiont uptake, while blocking TSR domains prevented symbiont uptake. Finally, the addition of exogenous TGFβ to TSR antibody-challenged anemones, reversed the blocking effect. These data suggest that the immune-suppressive TGFβ pathway is involved in early onset of the symbiosis. Since the TSR domain is implicated in the TGFβ pathway, these results support previous findings of the involvement of TGFβ in promoting tolerance of symbionts within the host. Apicomplexan parasites exploit scavenger receptor-TSR domain-binding to gain entry, and also use immune modulation to persist inside host cells. Data presented here suggest that dinoflagellates are utilizing the same mechanisms to form a mutualistic relationship with the cnidarian host. Overall, the work presented here provides new information about several cnidarian extracellular matrix proteins, with searches revealing large repertoires of both scavenger receptors and TSR-domain-containing proteins. Functional data suggest that both protein families are involved in the cnidarian-dinoflagellate symbiosis. Searches of the dinoflagellate genome did not find a clear dinoflagellate homologue to the apicomplexan TRAP proteins. However, this research provides further evidence that similar receptor-ligand interactions are involved in the entry of both beneficial and pathogenic microbes to host cells. These results add to growing knowledge about the complex molecular pathways that enable and support cnidarian-dinoflagellate symbiosis. An understanding of the mechanisms that support healthy symbiosis is essential when trying to predict the vitality and productivity of reef ecosystems in the face of climate change.</p>

520 Preliminary biomarker and pharmacodynamic (PD) activity of the TGFβ inhibitor SAR439459, alone or in combination with cemiplimab, in a phase 1 clinical study in patients with advanced solid tumors

BackgroundTransforming growth factor beta (TGFβ) is a bifunctional regulator of tumor growth playing a role in tumor immune evasion and resistance to checkpoint blockade. Increased activation of TGFβ pathway correlated with reduced overall survival in patients with PD-1 resistance/refractory tumors. Therefore, the combination of a TGFβ inhibitor with an anti-PD-1 agent may benefit patients who are resistant to checkpoint blockade. SAR439459 is a ”second generation” human anti-TGFβ IgG4 monoclonal antibody. Here we report the preliminary PD results and patient selection strategy (mesenchymal CRC) of SAR439459 ± anti-PD-1 cemiplimab in patients with advanced tumors from an on-going phase 1 study (NCT03192345).MethodsPeripheral blood, serum and tumor biopsies from patients were collected for the assessment of both predictive and PD biomarkers. A consensus molecular subtyping 4 (CMS4) gene classifier was developed and used to identify mesenchymal CRC tumors based on an in-silico experiment followed by a validation using ~200 procured CRC tumor biopsy samples with customized NanoString assay. TGFβ level in plasma and tumor was measured by ELISA to assess target engagement of SAR439459. Well-known immune modulation events as the PD readout were measured: 1) immunophenotyping of circulating immune cells ; 2) cytokine/chemokine production by MSD assay; 3) PD-L1, CD8+ T cells and FoxP3+ Tregs in tumor micro-environment (TME) by immunohistochemistry; 4) TGFβ pathway activation gene signature in TME by RNAseq.ResultsSAR439459 ± cemiplimab, induced inhibition of plasma TGFβ level ≥ 90% at doses ≥ 0.25mg/kg Q2W, together with a clear trend of decrease in intra-tumoral TGFβ. RNAseq data from paired biopsies revealed concomitant down-regulation of TGFβ pathway. In periphery, SAR439459 ± cemiplimab increased proliferating T and NK cells. Concomitantly, enhanced production of pro-inflammatory cytokines/chemokines confirmed peripheral immune activation. In TME, a trend of increased CD8+ T cell infiltration and conversion from ”immune-excluded” to ”immune-inflamed” phenotype was observed following the combination treatment in several cases. No significant modulation of PD-L1 or FoxP3 was observed from the available paired biopsies. Out of 137 pre-screened CRC patients, 58 (42%) were identified as carrying the CMS4 phenotype based on the gene classifier.ConclusionsClinical modulation of TGFβ level and the related pathway demonstrated SAR439459’s target engagement. Further analysis confirmed the peripheral immune activation in patients treated with SAR439459 ± cemiplimab. Coupled with CD8+ T cell modulation in TME, these findings suggest the identification of early PD biomarkers impacted by SAR439459 which is consistent with the mechanism of action and biological activity of TGFβ blockade therapy.Trial RegistrationNCT03192345Ethics ApprovalThe study protocols were approved by the institutional review board or independent ethics committee of each participating institution. All patients provided written informed consent priorto enrollment.ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

A human multi-lineage hepatic organoid model for liver fibrosis

To investigate the pathogenesis of a congenital form of hepatic fibrosis, human hepatic organoids were engineered to express the most common causative mutation for Autosomal Recessive Polycystic Kidney Disease (ARPKD). Here we show that these hepatic organoids develop the key features of ARPKD liver pathology (abnormal bile ducts and fibrosis) in only 21 days. The ARPKD mutation increases collagen abundance and thick collagen fiber production in hepatic organoids, which mirrors ARPKD liver tissue pathology. Transcriptomic and other analyses indicate that the ARPKD mutation generates cholangiocytes with increased TGFβ pathway activation, which are actively involved stimulating myofibroblasts to form collagen fibers. There is also an expansion of collagen-producing myofibroblasts with markedly increased PDGFRB protein expression and an activated STAT3 signaling pathway. Moreover, the transcriptome of ARPKD organoid myofibroblasts resemble those present in commonly occurring forms of liver fibrosis. PDGFRB pathway involvement was confirmed by the anti-fibrotic effect observed when ARPKD organoids were treated with PDGFRB inhibitors. Besides providing insight into the pathogenesis of congenital (and possibly acquired) forms of liver fibrosis, ARPKD organoids could also be used to test the anti-fibrotic efficacy of potential anti-fibrotic therapies.

TGFβ signalling is required to maintain pluripotency of human naïve pluripotent stem cells

The signalling pathways that maintain primed human pluripotent stem cells (hPSCs) have been well characterised, revealing a critical role for TGFβ/Activin/Nodal signalling. In contrast, the signalling requirements of naive human pluripotency have not been fully established. Here, we demonstrate that TGFβ signalling is required to maintain naive hPSCs. The downstream effector proteins – SMAD2/3 – bind common sites in naive and primed hPSCs, including shared pluripotency genes. In naive hPSCs, SMAD2/3 additionally bind to active regulatory regions near to naive pluripotency genes. Inhibiting TGFβ signalling in naive hPSCs causes the downregulation of SMAD2/3-target genes and pluripotency exit. Single-cell analyses reveal that naive and primed hPSCs follow different transcriptional trajectories after inhibition of TGFβ signalling. Primed hPSCs differentiate into neuroectoderm cells, whereas naive hPSCs transition into trophectoderm. These results establish that there is a continuum for TGFβ pathway function in human pluripotency spanning a developmental window from naive to primed states.

Canonical TGFβ Signaling and Its Contribution to Endometrial Cancer Development and Progression—Underestimated Target of Anticancer Strategies

Endometrial cancer is one of the leading gynecological cancers diagnosed among women in their menopausal and postmenopausal age. Despite the progress in molecular biology and medicine, no efficient and powerful diagnostic and prognostic marker is dedicated to endometrial carcinogenesis. The canonical TGFβ pathway is a pleiotropic signaling cascade orchestrating a variety of cellular and molecular processes, whose alterations are responsible for carcinogenesis that originates from different tissue types. This review covers the current knowledge concerning the canonical TGFβ pathway (Smad-dependent) induced by prototypical TGFβ isoforms and the involvement of pathway alterations in the development and progression of endometrial neoplastic lesions. Since Smad-dependent signalization governs opposed cellular processes, such as growth arrest, apoptosis, tumor cells growth and differentiation, as well as angiogenesis and metastasis, TGFβ cascade may act both as a tumor suppressor or tumor promoter. However, the final effect of TGFβ signaling on endometrial cancer cells depends on the cancer disease stage. The multifunctional role of the TGFβ pathway indicates the possible utilization of alterations in the TGFβ cascade as a potential target of novel anticancer strategies.

Genetic Modifiers and Phenotype of Duchenne Muscular Dystrophy: A Systematic Review and Meta-Analysis

The transforming growth factor beta (TGFβ) pathway could modulate the Duchenne muscular dystrophy (DMD) phenotype. This meta-analysis aims to estimate the association of genetic variants involved in the TGFβ pathway, including the latent transforming growth factor beta binding protein 4 (LTBP4) and secreted phosphoprotein 1 (SPP1) genes, among others, with age of loss of ambulation (LoA) and cardiac function in patients with DMD. Meta-analyses were conducted for the hazard ratio (HR) of LoA for each genetic variant. A subgroup analysis was performed in patients treated exclusively with glucocorticoids. Eight studies were included in the systematic review and four in the meta-analyses. The systematic review suggests a protective effect of LTBP4 haplotype IAAM (recessive model) for LoA. It is also suggested that the SPP1 rs28357094 genotype G (dominant model) is associated with early LoA in glucocorticoids-treated patients. The meta-analysis of the LTBP4 haplotype IAAM showed a protective association with LoA, with an HR = 0.78 (95% CI: 0.67–0.90). No association with LoA was observed for the SPP1 rs28357094. The LTBP4 haplotype IAAM is associated with a later LoA, especially in the Caucasian population, while the SPP1 rs28357094 genotype G could be associated with a poor response to glucocorticoids. Future research is suggested for SPP1 rs11730582, LTBP4 rs710160, and THBS1 rs2725797.

Reciprocal interplay between asporin and decorin: Implications in gastric cancer prognosis

Effective patient prognosis necessitates identification of novel tumor promoting drivers of gastric cancer (GC) which contribute to worsened conditions by analysing TCGA-gastric adenocarcinoma dataset. Small leucine-rich proteoglycans, asporin (ASPN) and decorin (DCN), play overlapping roles in development and diseases; however, the mechanisms underlying their interplay remain elusive. Here, we investigated the complex interplay of asporin, decorin and their interaction with TGFβ in GC tumor and corresponding normal tissues. The mRNA levels, protein expressions and cellular localizations of ASPN and DCN were analyzed using real-time PCR, western blot and immunohistochemistry, respectively. The protein-protein interaction was predicted by in-silico interaction analysis and validated by co-immunoprecipitation assay. The correlations between ASPN and EMT proteins, VEGF and collagen were achieved using western blot analysis. A significant increase in expression of ASPN in tumor tissue vs. normal tissue was observed in both TCGA and our patient cohort. DCN, an effective inhibitor of the TGFβ pathway, was negatively correlated with stages of GC. Co-immunoprecipitation demonstrated that DCN binds with TGFβ, in normal gastric epithelium, whereas in GC, ASPN preferentially binds TGFβ. Possible activation of the canonical TGFβ pathway by phosphorylation of SMAD2 in tumor tissues suggests its role as an intracellular tumor promoter. Furthermore, tissues expressing ASPN showed unregulated EMT signalling. Our study uncovers ASPN as a GC-promoting gene and DCN as tumor suppressor, suggesting that ASPN can act as a prognostic marker in GC. For the first time, we describe the physical interaction of TGFβ with ASPN in GC and DCN with TGFβ in GC and normal gastric epithelium respectively. This study suggests that prevention of ASPN-TGFβ interaction or overexpression of DCN could serve as promising therapeutic strategies for GC patients.