Mesenchymal-epithelial Cross-talk Research Articles

Cytokine signaling converging on IL11 in ILD fibroblasts provokes aberrant epithelial differentiation signatures.

Interstitial lung disease (ILD) is a heterogenous group of lung disorders where destruction and incomplete regeneration of the lung parenchyma often results in persistent architectural distortion of the pulmonary scaffold. Continuous mesenchyme-centered, disease-relevant signaling likely initiates and perpetuates the fibrotic remodeling process, specifically targeting the epithelial cell compartment, thereby destroying the gas exchange area. With the aim of identifying functional mediators of the lung mesenchymal-epithelial crosstalk with potential as new targets for therapeutic strategies, we developed a 3D organoid co-culture model based on human induced pluripotent stem cell-derived alveolar epithelial type 2 cells that form alveolar organoids in presence of lung fibroblasts from fibrotic-ILD patients, in our study referring to cases of pulmonary fibrosis, as well as control cell line (IMR-90). While organoid formation capacity and size was comparable in the presence of fibrotic-ILD or control lung fibroblasts, metabolic activity was significantly increased in fibrotic-ILD co-cultures. Alveolar organoids cultured with fibrotic-ILD fibroblasts further demonstrated reduced stem cell function as reflected by reduced Surfactant Protein C gene expression together with an aberrant basaloid-prone differentiation program indicated by elevated Cadherin 2, Bone Morphogenic Protein 4 and Vimentin transcription. To screen for key mediators of the misguided mesenchymal-to-epithelial crosstalk with a focus on disease-relevant inflammatory processes, we used mass spectrometry and characterized the secretome of end stage fibrotic-ILD lung fibroblasts in comparison to non-chronic lung disease (CLD) patient fibroblasts. Out of the over 2000 proteins detected by this experimental approach, 47 proteins were differentially abundant comparing fibrotic-ILD and non-CLD fibroblast secretome. The fibrotic-ILD secretome profile was dominated by chemokines, including CXCL1, CXCL3, and CXCL8, interfering with growth factor signaling orchestrated by Interleukin 11 (IL11), steering fibrogenic cell-cell communication, and proteins regulating extracellular matrix remodeling including epithelial-to-mesenchymal transition. When in turn treating alveolar organoids with IL11, we recapitulated the co-culture results obtained with primary fibrotic-ILD fibroblasts including changes in metabolic activity. We identified mediators likely contributing to the disease-perpetuating mesenchymal-to-epithelial crosstalk in ILD. In our alveolar organoid co-cultures, we were able to highlight the importance of fibroblast-initiated aberrant epithelial differentiation and confirmed IL11 as a key player in fibrotic-ILD pathogenesis by unbiased fibroblast secretome analysis.

A26 BMP-SIGNALING IMPAIRED TELOCYTES CREATE A DISRUPTED NICHE GRADIENT FOSTERING COLITIS-ASSOCIATED CANCER

Abstract Background The colonic stem cell niche is established by a gradient of WNT, R-spondin, BMP factors and their antagonists along the colonic epithelial vertical axis. Telocytes (TCFoxL1+) are mesenchymal cell forming a 3D hub underneath the epithelium, identified as an important source of niche factors. Specifically, they express non-canonical (nc) WNT factors and are the richest source of BMPs. Disruption of the BMPs gradient has been shown to be related to the development of several gastrointestinal diseases like Inflammatory Bowel Diseases (IBD). Such chronic inflammation drives the onset of Colitis-Associated Cancer (CAC) in about 60% of IBD patients. We previously showed that following a chronic inflammatory stress, 50% of the KO mouse for the BMP receptor 1a in colon telocytes (Bmpr1a△FoxL1+) presented malignant epithelial transformations. These cancer-like regions showed an aberrant epithelial b-catenin localization and an enlargement of the double positive α-SMA+/Vimentin+ mesenchymal population. Purpose: Loss of BMP signaling in TCFoxL1+ affects the mesenchymal-epithelial crosstalk and makes the colonic epithelium vulnerable to injuries, promoting/perpetuating inflammation fostering CAC onset. Method Following a DSS-based chronic inflammatory challenge in mutant and control mice, TCFoxL1+ ultrastructure was analyzed using transmission electron microscopy. Expression levels of members of the WNT-BMP axis (BMPs, WNTs and associated antagonists) were evaluated by qPCR in tumor-like areas and adjacent tissue. YAP cellular localization was evaluated by immunofluorescence in colon after chronic DSS challenge in Bmpr1a△FoxL1+ mice and controls. To differentiate cancer-associated fibroblasts (CAFs) subtypes, myCAF (myofibroblastic) and iCAF (inflammatory), in tumor-like region and adjacent tissue, we used co-staining against gp38, ICAM, Tagln and αSMA. Result(s) Following a chronic DSS-challenge, electron microscopy analysis demonstrated that TCFoxL1+ in the control mice exhibited a shortening and erosion in their telopodes (Tp). TCFoxL1+ in Bmpr1a△FoxL1+ mice tumor-like regions presented an expanded endoplasmic reticulum with fragmented and dilated Tp. A significant increase in BMP 4, 5 and 7 and in Wnt5 (nc) was detected in Bmpr1a△FoxL1+ mice compared to controls. Confocal analysis revealed a strong nuclear accumulation of YAP in cancer-like regions in mutant mice compared to controls. Finally, tumour-like regions presented an heterogeneous distribution of iCAF and myCAF compared to controls. Conclusion(s) These results exposed that the disruption of TCFoxL1+ associated BMP signaling disturbs the WNT-BMP gradient essential for the optimal maintenance of the SC niche and thus impacting epithelial regeneration when under stress. Thus, defective TCFoxL1+ assume a key role in the poor regeneration process of the epithelium which in the end promotes the development and progression of CAC. Please acknowledge all funding agencies by checking the applicable boxes below CIHR Disclosure of Interest None Declared

Mesenchymal-epithelial crosstalk shapes intestinal regionalisation via Wnt and Shh signalling

Organs are anatomically compartmentalised to cater for specialised functions. In the small intestine (SI), regionalisation enables sequential processing of food and nutrient absorption. While several studies indicate the critical importance of non-epithelial cells during development and homeostasis, the extent to which these cells contribute to regionalisation during morphogenesis remains unexplored. Here, we identify a mesenchymal-epithelial crosstalk that shapes the developing SI during late morphogenesis. We find that subepithelial mesenchymal cells are characterised by gradients of factors supporting Wnt signalling and stimulate epithelial growth in vitro. Such a gradient impacts epithelial gene expression and regional villus formation along the anterior-posterior axis of the SI. Notably, we further provide evidence that Wnt signalling directly regulates epithelial expression of Sonic Hedgehog (SHH), which, in turn, acts on mesenchymal cells to drive villi formation. Taken together our results uncover a mechanistic link between Wnt and Hedgehog signalling across different cellular compartments that is central for anterior-posterior regionalisation and correct formation of the SI.

Elevated Protein Kinase A Activity in Stomach Mesenchyme Disrupts Mesenchymal-epithelial Crosstalk and Induces Preneoplasia.

Background & AimsMesenchymal-epithelial crosstalk (MEC) in the stomach is executed by pathways such as bone morphogenetic protein (BMP) and extracellular signal-regulated kinase (ERK). Mis-regulation of MEC disrupts gastric homeostasis and causes tumorigenesis. Protein Kinase A (PKA) crosstalks with BMP and ERK signaling; however, PKA function(s) in stomach development and homeostasis remains undefined.MethodsWe generated a novel Six2-Cre+/-PKAcαRfl/wt (CA-PKA) mouse in which expression of constitutive-active PKAcαR was induced in gastric mesenchyme progenitors. Lineage tracing determined spatiotemporal activity of Six2-Cre in the stomach. For phenotyping CA-PKA mice histological, co-immunofluorescence, immunoblotting, mRNA sequencing, and bioinformatics analyses were performed.ResultsLineage tracing showed that Six2-Cre activity in the stomach is restricted to the mesenchymal compartment. CA-PKA mice showed disruption of gastric homeostasis characterized by aberrant mucosal development and epithelial hyperproliferation; ultimately developing multiple features of gastric corpus preneoplasia including decreased parietal cells, mucous cell hyperplasia, spasmolytic peptide expressing metaplasia with intestinal characteristics, and dysplastic and invasive cystic glands. Furthermore, mutant corpus showed marked chronic inflammation characterized by infiltration of lymphocytes and myeloid-derived suppressor cells along with the upregulation of innate and adaptive immune system components. Striking upregulation of inflammatory mediators and STAT3 activation was observed. Mechanistically, we determined there is an activation of ERK1/2 and downregulation of BMP/SMAD signaling characterized by marked upregulation of BMP inhibitor gremlin 1.ConclusionsWe report a novel role of PKA signaling in gastric MEC execution and show that PKA activation in the gastric mesenchyme drives preneoplasia by creating a proinflammatory and proproliferative microenvironment associated with the downregulation of BMP/SMAD signaling and activation of ERK1/2.

Mesenchymal Niches for Digestive Organ Development, Homeostasis, and Disease.

Mesenchymal-epithelial crosstalk plays a crucial role in organ development and stem cell function. However, the identity of the mesenchymal cells involved in this exchange was unclear. Recent significant advances in single-cell transcriptomics have defined the heterogeneity of these mesenchymal niches. By combining multiomic profiling, animal models, and organoid culture, new studies have not only demonstrated the roles of diverse mesenchymal cell populations but also defined the mechanisms underlying their regulation of niche signals. Focusing on several digestive organs, we describe how similar and diverse mesenchymal cell populations promote organ development and maintain proper stem cell activity, and how the heterogeneity of mesenchymal niches is altered in digestive diseases such as inflammation and cancer.

Noncanonical WNT-5A signaling impairs endogenous lung repair in COPD.

Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide. One main pathological feature of COPD is the loss of functional alveolar tissue without adequate repair (emphysema), yet the underlying mechanisms are poorly defined. Reduced WNT-β-catenin signaling is linked to impaired lung repair in COPD; however, the factors responsible for attenuating this pathway remain to be elucidated. Here, we identify a canonical to noncanonical WNT signaling shift contributing to COPD pathogenesis. We demonstrate enhanced expression of noncanonical WNT-5A in two experimental models of COPD and increased posttranslationally modified WNT-5A in human COPD tissue specimens. WNT-5A was increased in primary lung fibroblasts from COPD patients and induced by COPD-related stimuli, such as TGF-β, cigarette smoke (CS), and cellular senescence. Functionally, mature WNT-5A attenuated canonical WNT-driven alveolar epithelial cell wound healing and transdifferentiation in vitro. Lung-specific WNT-5A overexpression exacerbated airspace enlargement in elastase-induced emphysema in vivo. Accordingly, inhibition of WNT-5A in vivo attenuated lung tissue destruction, improved lung function, and restored expression of β-catenin-driven target genes and alveolar epithelial cell markers in the elastase, as well as in CS-induced models of COPD. We thus identify a novel essential mechanism involved in impaired mesenchymal-epithelial cross talk in COPD pathogenesis, which is amenable to therapy.

Thymic Mesenchymal Cells Have a Distinct Transcriptomic Profile

In order to understand the role of mesenchymal cells (MCs) in the adult thymus, we performed whole transcriptome analyses of primary thymic, bone, and skin MCs. These three MC populations shared expression of 2850 core MC genes involved in generic processes including interactions with tissue-resident macrophages. Moreover, we discovered that 2036 genes were differentially expressed, by at least 5-fold, in the three MC populations. Genes preferentially expressed in thymic MCs are instrumental in clearance of apoptotic thymocytes by macrophages, maintenance of a noninflammatory milieu, and attraction-expansion of thymocyte progenitors. Thymic and bone MCs share other sets of differentially expressed genes implicated in resolution of inflammation and expansion of hematolymphoid progenitors. Consistent with the fact that thymic and skin MCs have to support epithelial cells, they express at higher levels genes mediating epithelial cell adhesion to basement membrane and mesenchymal-epithelial cross-talk. Differentially expressed genes preferentially expressed by bone MCs are connected to formation and remodeling of bone, whereas those preferentially expressed in skin MCs are involved in skin and hair follicle homeostasis. We conclude that MCs from different organs display substantial heterogeneity and that the transcriptome of thymic MCs is exquisitely suited for interactions with epithelial and hematolymphoid cells in an environment with a high apoptosis rate.

Commentary: A Breath of Fresh Air on the Mesenchyme: Impact of Impaired Mesenchymal Development on the Pathogenesis of Bronchopulmonary Dysplasia.

Commentary: A Breath of Fresh Air on the Mesenchyme: Impact of Impaired Mesenchymal Development on the Pathogenesis of Bronchopulmonary Dysplasia.

Sox2 in the Dermal Papilla Niche Controls Hair Growth by Fine-Tuning BMP Signaling in Differentiating Hair Shaft Progenitors

How dermal papilla (DP) niche cells regulate hair follicle progenitors to control hair growth remains unclear. Using Tbx18(Cre) to target embryonic DP precursors, we ablate the transcription factor Sox2 early and efficiently, resulting in diminished hair shaft outgrowth. We find that DP niche expression of Sox2 controls the migration speed of differentiating hair shaft progenitors. Transcriptional profiling of Sox2 null DPs reveals increased Bmp6 and decreased BMP inhibitor Sostdc1, a direct Sox2 transcriptional target. Subsequently, we identify upregulated BMP signaling in knockout hair shaft progenitors and demonstrate that Bmp6 inhibits cell migration, an effect that can be attenuated by Sostdc1. A shorter and Sox2-negative hair type lacks Sostdc1 in the DP and shows reduced migration and increased BMP activity of hair shaft progenitors. Collectively,our data identify Sox2 as a key regulator of hair growth that controls progenitor migration by fine-tuning BMP-mediated mesenchymal-epithelial crosstalk.

Endometrial stromal beta-catenin is required for steroid-dependent mesenchymal-epithelial cross talk and decidualization

BackgroundBeta-catenin is part of a protein complex associated with adherens junctions. When allowed to accumulate to sufficient levels in its dephosphorylated form, beta-catenin serves as a transcriptional co-activator associated with a number of signaling pathways, including steroid hormone signaling pathways.MethodsTo investigate the role of beta-catenin in progesterone (P4) signaling and female reproductive physiology, conditional ablation of Ctnnb1 from the endometrial mesenchymal (i.e. stromal and myometrial), but not epithelial, compartment was accomplished using the Amhr2-Cre mice. Experiments were conducted to assess the ability of mutant female mice to undergo pregnancy and pseudopregnancy by or through oil-induced decidualization. The ability of uteri from mutant female mice to respond to estrogen (E2) and P4 was also determined.ResultsConditional deletion of Ctnnb1 from the mesenchymal compartment of the uterus resulted in infertility stemming, in part, from complete failure of the uterus to decidualize. E2-stimulated epithelial cell mitosis and edematization were not altered in mutant uteri indicating that the mesenchyme is capable of responding to E2. However, exposure of ovariectomized mutant female mice to a combined E2 and P4 hormone regimen consistent with early pregnancy revealed that mesenchymal beta-catenin is essential for indirectly opposing E2-induced epithelial proliferation by P4 and in some mice resulted in development of endometrial metaplasia. Lastly, beta-catenin is also required for the induced expression of genes that are known to play a fundamental role in decidualization such as Ihh, Ptch1, Gli1 and Muc1ConclusionsThree salient points derive from these studies. First, the findings demonstrate a mechanistic linkage between the P4 and beta-catenin signaling pathways. Second, they highlight an under appreciated role for the mesenchymal compartment in indirectly mediating P4 signaling to the epithelium, a process that intimately involves mesenchymal beta-catenin. Third, the technical feasibility of deleting genes in the mesenchymal compartment of the uterus in an effort to understand decidualization and post-natal interactions with the overlying epithelium has been demonstrated. It is concluded that beta-catenin plays an integral role in selective P4-directed epithelial-mesenchymal communication in both the estrous cycling and gravid uterus.

Transcriptome analysis of embryonic mammary cells reveals insights into mammary lineage establishment

IntroductionThe mammary primordium forms during embryogenesis as a result of inductive interactions between its constitutive tissues, the mesenchyme and epithelium, and represents the earliest evidence of commitment to the mammary lineage. Previous studies of embryonic mouse mammary epithelium indicated that, by mid-gestation, these cells are determined to a mammary cell fate and that a stem cell population has been delimited. Mammary mesenchyme can induce mammary development from simple epithelium even across species and classes, and can partially restore features of differentiated tissue to mouse mammary tumours in co-culture experiments. Despite these exciting properties, the molecular identity of embryonic mammary cells remains to be fully characterised.MethodsHere, we define the transcriptome of the mammary primordium and the two distinct cellular compartments that comprise it, the mammary primordial bud epithelium and mammary mesenchyme. Pathway and network analysis was performed and comparisons of embryonic mammary gene expression profiles to those of both postnatal mouse and human mammary epithelial cell sub-populations and stroma were made.ResultsSeveral of the genes we have detected in our embryonic mammary cell signatures were previously shown to regulate mammary cell fate and development, but we also identified a large number of novel candidates. Additionally, we determined genes that were expressed by both embryonic and postnatal mammary cells, which represent candidate regulators of mammary cell fate, differentiation and progenitor cell function that could signal from mammary lineage inception during embryogenesis through postnatal development. Comparison of embryonic mammary cell signatures with those of human breast cells identified potential regulators of mammary progenitor cell functions conserved across species.ConclusionsThese results provide new insights into genetic regulatory mechanisms of mammary development, particularly identification of novel potential regulators of mammary fate and mesenchymal-epithelial cross-talk. Since cancers may represent diseases of mesenchymal-epithelial communications, we anticipate these results will provide foundations for further studies into the fundamental links between developmental, stem cell and breast cancer biology.

Stromal Hoxa5 function controls the growth and differentiation of mammary alveolar epithelium

Recent progress has enlightened the involvement of Hox genes in organogenesis. Several Hox genes are expressed in normal and neoplastic mammary glands. Using Hoxa5 mutant mice, we showed that Hoxa5-/- females present nursing defects. Characterization of the Hoxa5-/- mammary gland phenotype reveals changes in proliferation and differentiation of the epithelium of nulliparous and pregnant Hoxa5-/- females that precede the abnormal secretory activity at parturition. These defects likely underlie the incapacity of Hoxa5-/- dams to properly feed their pups. Hoxa5 expression is restricted to the mammary stroma at specific stages of mammary gland development. The loss of Hoxa5 function causes accelerated lobuloalveolar epithelium development, a phenotype that can be rescued upon grafting of mutant mammary epithelium into wild-type fat pads. Conversely, reciprocal grafting experiments demonstrate that Hoxa5-/- stroma cannot support normal proliferation of wild-type mammary epithelium. These data establish the essential contribution of Hoxa5 to mammary epithelium instruction by means of mesenchymal-epithelial crosstalk.

Bone marrow stromal cells, preadipocytes, and dermal fibroblasts promote epidermal regeneration in their distinctive fashions.

Mesenchymal cell types, under mesenchymal-epithelial interaction, are involved in tissue regeneration. Here we show that bone marrow stromal cells (BMSCs), subcutaneous preadipocytes, and dermal fibroblasts distinctively caused keratinocytes to promote epidermal regeneration, using a skin reconstruction model by their coculture with keratinocytes. Three mesenchymal cell types promoted the survival, growth, and differentiation of keratinocytes, whereas BMSCs and preadipocytes inhibited their apoptosis. BMSCs and preadipocytes induced keratinocytes to reorganize rete ridge- and epidermal ridge-like structures, respectively. Keratinocytes with fibroblasts or BMSCs expressed the greatest amount of interleukin (IL)-1alpha protein, which is critical for mesenchymal-epithelial cross-talk in skin. Keratinocytes with or without three mesenchymal supports displayed another cross-talk molecule, c-Jun protein. Without direct mesenchymal-epithelial contact, the rete ridge- and epidermal ridge-like structures were not replicated, whereas the other phenomena noted above were. DNA microarray analysis showed that the mesenchymal-epithelial interaction affected various gene expressions of keratinocytes and mesenchymal cell types. Our results suggest that not only skin-localized fibroblasts and preadipocytes but also BMSCs accelerate epidermal regeneration in complexes and that direct contact between keratinocytes and BMSCs or preadipocytes is required for the skin-specific morphogenesis above, through mechanisms that differ from the IL-1alpha/c-Jun pathway.

Gastric stem cells: an update.

Cells of the gastric mucosa undergo constant renewal, the rate depending on the health of the tissue (inflammation, ulceration, carcinogenesis). While much attention has been focused on the mechanism of mucosal damage and the pathogenesis of ulceration, there has recently been the recognition that elucidation of the nature of the gastric stem cell lineages as well as the regulators of phenotype expression of this system may yield considerable biological information as well as open the door to the identification of areas of therapeutic relevance. Chimeric and X-inactivation studies in mice and humans demonstrate that each region of the gastric mucosa is morphologically diverse (antrum is different to the fundus), with its own repertoire of cell types and glandular structures. The current evidence suggests that a single stem cell in every gastric gland indirectly gives rise to a clone of all differentiated cells, by production of committed progenitor cells. It is also this multipotential cell that produces new crypts by crypt fission, repairs entire crypts when damaged, and gives rise to the ulcer-associated cell lineage and gastric carcinomas. It is likely that this stem cell occupies a niche in the isthmus composed of mesenchymal cells and extracellular matrix factors, which regulates the function of the cell via mesenchymal-epithelial cross talk. The molecular events (IGF-signaling) that regulate the development of the gastric gland in the mice have begun to be understood. Ultimately, the identification of these pathways will play an important role in identifying new molecular targets for the treatment of gastric disease.

Function of AP-1 target genes in mesenchymal–epithelial cross-talk in skin

An increasing number of examples on the importance of mesenchymal–epithelial interactions in physiological (e.g. embryonic development) and pathological (tumourigenesis) processes have been described. This is best illustrated in the skin, where the well-controlled balance of keratinocyte proliferation and differentiation forms the basis for a proper histoarchitecture of the epidermis. Here, a double paracrine loop of cytokines, which are synthesised and secreted by cells of the epidermis (keratinocytes) and the underlying dermis (fibroblasts) seems to play a major role. The aim of this commentary is to review research that has investigated the role of specific subunits of transcription factor AP-1 (Jun/Fos) in this regulatory network. Using an in vitro skin equivalent model strong evidence was provided for a critical and specific function of c-Jun and JunB in mesenchymal–epithelial interaction in the skin by regulating the expression of interleukin-1 (IL-1)-induced keratinocyte growth factor (KGF) and GM-CSF in fibroblasts. These factors, in turn, adjust the balance between proliferation and differentiation of keratinocytes ensuring proper architecture of the epidermis. This commentary will summarise our current knowledge on the molecular mechanisms underlying AP-1-dependent mesenchymal–epithelial interactions and discuss the physiological relevance of these in vitro findings in skin physiology and pathology.