SM22α deficiency: promoting vascular fibrosis via SRF-SMAD3-mediated activation of Col1a2 transcription following arterial injury.

Vascular fibrosis, characterized by increased Type I collagen expression, significantly contributes to vascular remodeling. Our previous studies show that disrupting the expression of SM22α (aka SM22, Tagln) induces extensive vascular remodeling following arterial injury, involving oxidative stress, inflammation, and chondrogenesis within the vessel wall. This study aims to investigate the molecular mechanisms underlying the transcription of Col1a2, a key fibrotic extracellular matrix marker. We observed upregulation of COL1A2 in the arterial wall of Sm22-/- mice following carotid injury. Bioinformatics and molecular analyses reveal that Col1a2 transcription depends on a CArG box in the promoter, activated synergistically by SRF and SMAD3. Notably, we detected enhanced nuclear translocation of both SRF and SMAD3 in the smooth muscle cells of the injured carotid artery in Sm22-/- mice. These findings demonstrate that SM22 deficiency regulates vascular fibrosis through the interaction of SRF and the SMAD3-mediated canonical TGF-ß1 signal pathway, suggesting SM22α as a potential therapeutic target for preventing vascular fibrosis.

Reduced Neurog3 Gene Dosage Shifts Enteroendocrine Progenitor Towards Goblet Cell Lineage in the Mouse Intestine.

BACKGROUND & AIMS: Transient expression of Neurog3 commits intestinal secretory progenitors to become enteroendocrine-biased progenitors and hence drive enteroendocrine differentiation. Loss of Neurog3 in mouse resulted in the depletion of intestinal enteroendocrine cells (EECs) and an increase in goblet cells. Earlier studies in developing mouse pancreas identified a role of Neurog3 gene dosage in endocrine and exocrine cell fate allocation. We aimed to determine whether Neurog3 gene dosage controls fate choice of enteroendocrine progenitors. METHODS: We acquired mutant Neurog3 reporter mice carrying 2, 1, or null Neurog3 alleles to study Neurog3 gene dosage effect by lineage tracing. Cell types arising from Neurog3+ progenitors were determined by immunohistochemistry using antibodies against intestinal lineage-specific markers. RNA sequencing of sorted Neurog3+/+, Neurog3+/-, or bulk intestinal cells were performed and differentially expressed genes were analyzed. RESULTS: We identified 2731 genes enriched in sorted Neurog3+/+-derived cells in the Neurog3+/+EYFP mouse intestine when compared with bulk duodenum epithelial cells. In the intestine of Neurog3+/-EGFP heterozygous mouse, we observed a 63% decrease in EEC numbers. Many Neurog3-derived cells stained for goblet marker Mucin 2. RNA sequencing of sorted Neurog3+/- cells uncovered enriched expression of genes characteristic for both goblet and enteroendocrine cells, indicating the mixed lineages arose from Neurog3+ progenitors. Consistent with this hypothesis, deletion of both Neurog3 alleles resulted in the total absence of EECs. All Neurog3+-derived cells stained for Mucin 2. CONCLUSIONS: We identified that the fate of Neurog3+ enteroendocrine progenitors is dependent on Neurog3 gene dosage. High Neurog3 gene dosage enforces the commitment of secretory progenitors to an EE lineage, while constraining their goblet cell lineage potential. Transcriptome profiling data was deposited to Gene Ontology omnibus, accession number: GSE149203.

Intestinal Neurod1 expression impairs paneth cell differentiation and promotes enteroendocrine lineage specification.

Transcription factor Neurod1 is required for enteroendocrine progenitor differentiation and maturation. Several earlier studies indicated that ectopic expression of Neurod1 converted non- neuronal cells into neurons. However, the functional consequence of ectopic Neurod1 expression has not been examined in the GI tract, and it is not known whether Neurod1 can similarly switch cell fates in the intestine. We generated a mouse line that would enable us to conditionally express Neurod1 in intestinal epithelial cells at different stages of differentiation. Forced expression of Neurod1 throughout intestinal epithelium increased the number of EECs as well as the expression of EE specific transcription factors and hormones. Furthermore, we observed a substantial reduction of Paneth cell marker expression, although the expressions of enterocyte-, tuft- and goblet-cell specific markers are largely not affected. Our earlier study indicated that Neurog3+ progenitor cells give rise to not only EECs but also Goblet and Paneth cells. Here we show that the conditional expression of Neurod1 restricts Neurog3+ progenitors to adopt Paneth cell fate, and promotes more pronounced EE cell differentiation, while such effects are not seen in more differentiated Neurod1+ cells. Together, our data suggest that forced expression of Neurod1 programs intestinal epithelial cells more towards an EE cell fate at the expense of the Paneth cell lineage and the effect ceases as cells mature to EE cells.

Mechanosensitive ion channel Piezo2 is important for enterochromaffin cell response to mechanical forces.

KEY POINTS: The gastrointestinal epithelial enterochromaffin (EC) cell synthesizes the vast majority of the body's serotonin. As a specialized mechanosensor, the EC cell releases this serotonin in response to mechanical forces. However, the molecular mechanism of EC cell mechanotransduction is unknown. In the present study, we show, for the first time, that the mechanosensitive ion channel Piezo2 is specifically expressed by the human and mouse EC cells. Activation of Piezo2 by mechanical forces results in a characteristic ionic current, the release of serotonin and stimulation of gastrointestinal secretion. Piezo2 inhibition by drugs or molecular knockdown decreases mechanosensitive currents, serotonin release and downstream physiological effects. The results of the present study suggest that the mechanosensitive ion channel Piezo2 is specifically expressed by the EC cells of the human and mouse small bowel and that it is important for EC cell mechanotransduction. ABSTRACT: The enterochromaffin (EC) cell in the gastrointestinal (GI) epithelium is the source of nearly all systemic serotonin (5-hydroxytryptamine; 5-HT), which is an important neurotransmitter and endocrine, autocrine and paracrine hormone. The EC cell is a specialized mechanosensor, and it is well known that it releases 5-HT in response to mechanical forces. However, the EC cell mechanotransduction mechanism is unknown. The present study aimed to determine whether Piezo2 is involved in EC cell mechanosensation. Piezo2 mRNA was expressed in human jejunum and mouse mucosa from all segments of the small bowel. Piezo2 immunoreactivity localized specifically within EC cells of human and mouse small bowel epithelium. The EC cell model released 5-HT in response to stretch, and had Piezo2 mRNA and protein, as well as a mechanically-sensitive inward non-selective cation current characteristic of Piezo2. Both inward currents and 5-HT release were inhibited by Piezo2 small interfering RNA and antagonists (Gd3+ and D-GsMTx4). Jejunum mucosal pressure increased 5-HT release and short-circuit current via submucosal 5-HT3 and 5-HT4 receptors. Pressure-induced secretion was inhibited by the mechanosensitive ion channel antagonists gadolinium, ruthenium red and D-GsMTx4. We conclude that the EC cells in the human and mouse small bowel GI epithelium selectively express the mechanosensitive ion channel Piezo2, and also that activation of Piezo2 by force leads to inward currents, 5-HT release and an increase in mucosal secretion. Therefore, Piezo2 is critical to EC cell mechanosensitivity and downstream physiological effects.

Besides an ITIM/SHP-1-dependent pathway, CD22 collaborates with Grb2 and plasma membrane calcium-ATPase in an ITIM/SHP-1-independent pathway of attenuation of Ca2+i signal in B cells.

CD22 is a surface immunoglobulin implicated in negative regulation of B cell receptor (BCR) signaling; particularly inhibiting intracellular Ca2+ (Ca2+i)signals. Its cytoplasmic tail contains six tyrosine residues (Y773/Y783/Y817/Y828/Y843/Y863, designated Y1~Y6 respectively), including three (Y2/5/6) lying within immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that serve to recruit the protein tyrosine phosphatase SHP-1 after BCR activation-induced phosphorylation. The mechanism of inhibiting Ca2+i by CD22 has been poorly understood. Previous study demonstrated that CD22 associated with plasma membrane calcium-ATPase (PMCA) and enhanced its activity (Chen, J. et al. Nat Immunol 2004;5:651-7). The association is dependent on BCR activation-induced cytoplasmic tyrosine phosphorylation, because CD22 with either all six tyrosines mutated to phenylalanines or cytoplasmic tail truncated loses its ability to associate with PMCA. However, which individual or a group of tyrosine residues determine the association and how CD22 and PMCA interacts, are still unclear. In this study, by using a series of CD22 tyrosine mutants, we found that ITIM Y2/5/6 accounts for 34.3~37.1% Ca2+i inhibition but is irrelevant for CD22/PMCA association. Non-ITIM Y4 and its YEND motif contribute to the remaining 69.4~71.7% Ca2+i inhibition and is the binding site for PMCA-associated Grb2. Grb2, independently of BCR cross-linking, is constitutively associated with and directly binds to PMCA in both chicken and human B cells. Knockout of Grb2 by CRISPR/Cas9 completely disrupted the CD22/PMCA association. Thus, our results demonstrate for the first time that in addition to previously-identified ITIM/SHP-1-dependent pathway, CD22 holds a major pathway of negative regulation of Ca2+i signal, which is ITIM/SHP-1-independent, but Y4/Grb2/PMCA-dependent.

Distinct cellular origins for serotonin-expressing and enterochromaffin-like cells in the gastric corpus.

BACKGROUND & AIMS: The alimentary tract contains a diffuse endocrine system comprising enteroendocrine cells that secrete peptides or biogenic amines to regulate digestion, insulin secretion, food intake, and energy homeostasis. Lineage analysis in the stomach revealed that a significant fraction of endocrine cells in the gastric corpus did not arise from Neurogenin3 (Neurog3)-expressing cells, unlike enteroendocrine cells elsewhere in the digestive tract. We aimed to isolate enriched serotonin-secreting and enterochromaffin-like (ECL) cells from the stomach and to clarify their cellular origin. METHODS: We used Neurogenic differentiation 1 (NeuroD1) and Neurog3 lineage analysis and examined the differentiation of serotonin-producing and ECL cells in stomach tissues of NeuroD1-cre;ROSA(tdTom), tryptophan hydroxylase 1 (Tph1)-cyan fluorescent protein (CFP), c-Kit(wsh/wsh), and Neurog3Cre;ROSA(tdTom) mice by immunohistochemistry. We used fluorescence-activated cell sorting to isolate each cell type for gene expression analysis. We also performed RNA sequencing analysis of ECL cells. RESULTS: Neither serotonin-secreting nor ECL cells of the corpus arose from cells expressing NeuroD1. Serotonin-secreting cells expressed a number of mast cell genes but not genes associated with endocrine differentiation; they did not develop in c-Kit(wsh/wsh) mice and were labeled with transplanted bone marrow cells. RNA sequencing analysis of ECL cells revealed high expression levels of many genes common to endocrine cells, including transcription factors, hormones, ion channels, and solute transporters but not markers of bone marrow cells. CONCLUSIONS: Serotonin-expressing cells of the gastric corpus of mice appear to be bone marrow-derived mucosal mast cells. Gene expression analysis of ECL cells indicated that they are endocrine cells of epithelial origin that do not express the same transcription factors as their intestinal enteroendocrine cell counterparts.

Notch signaling differentially regulates the cell fate of early endocrine precursor cells and their maturing descendants in the mouse pancreas and intestine.

Notch signaling inhibits differentiation of endocrine cells in the pancreas and intestine. In a number of cases, the observed inhibition occurred with Notch activation in multipotential cells, prior to the initiation of endocrine differentiation. It has not been established how direct activation of Notch in endocrine precursor cells affects their subsequent cell fate. Using conditional activation of Notch in cells expressing Neurogenin3 or NeuroD1, we examined the effects of Notch in both organs, on cell fate of early endocrine precursors and maturing endocrine-restricted cells, respectively. Notch did not preclude the differentiation of a limited number of endocrine cells in either organ when activated in Ngn3(+) precursor cells. In addition, in the pancreas most Ngn3(+) cells adopted a duct but not acinar cell fate; whereas in intestinal Ngn3(+) cells, Notch favored enterocyte and goblet cell fates, while selecting against endocrine and Paneth cell differentiation. A small fraction of NeuroD1(+) cells in the pancreas retain plasticity to respond to Notch, giving rise to intraislet ductules as well as cells with no detectable pancreatic lineage markers that appear to have limited ultrastructural features of both endocrine and duct cells. These results suggest that Notch directly regulates cell fate decisions in multipotential early endocrine precursor cells. Some maturing endocrine-restricted NeuroD1(+) cells in the pancreas switch to the duct lineage in response to Notch, indicating previously unappreciated plasticity at such a late stage of endocrine differentiation.

RIF-1, a novel nuclear receptor corepressor that associates with the nuclear matrix.

The retinoic acid receptors (RARs) are ligand-dependent transcription factors that play critical roles in cell differentiation, embryonic development, and tumor suppression. RAR transcriptional activities are mediated by a growing family of nuclear receptor (NR) coregulators. Here we report the cloning and characterization of a novel protein RIF1 (receptor interacting factor) that interacts with RARalpha in vivo and in vitro. RIF1 encodes a novel 739 amino acid protein that is ubiquitously expressed in a variety of tissues and cell lines. GST-pull down assays show that RIF1 also interacts with a number of other NRs. The interaction domain of RIF1 for RARalpha is located at the C-terminal region of RIF1, between amino acids 512 and 674. RIF1 is localized exclusively in the cell nucleus and specifically to the nuclear matrix. Mutation of the nuclear localization signal abolishes this nuclear localization and causes RIF1 to appear in the cytoplasm. Co-transfection of RIF1 with RAR causes RAR to localize to the nuclear matrix. RIF1 contains a strong transcriptional repression domain that robustly inhibits ligand-dependent transcriptional activation by RARalpha. This domain is located to the distal C-terminal 100 amino acids, distinct from the RARalpha-interaction and nuclear matrix-targeting domains. The transcriptional repression activity of RIF1 is mediated at least in part through direct recruitment of histone deacetylases. This study identifies RIF1 as a novel nuclear matrix transcription repressor, and suggests a potential role of RIF1 that regulates NR transcriptional activity.

Steroid receptor coactivator 3 is a coactivator for myocardin, the regulator of smooth muscle transcription and differentiation.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) constitutes a key event in atherosclerosis, neointimal hyperplasia, and the response to vascular injury. Estrogen receptor alpha (ERalpha) mediates the protective effects of estrogen in injured blood vessels and regulates ligand-dependent gene expression in vascular cells. However, the molecular mechanisms mediating ERalpha-dependent VSMC gene expression and VSMC proliferation after vascular injury are not well defined. Here, we report that the ER coactivator steroid receptor coactivator 3 (SRC3) is also a coactivator for the major VSMC transcription factor myocardin, which is required for VSMC differentiation to the nonproliferative, contractile state. The N terminus of SRC3, which contains a basic helix-loop-helix/Per-ARNT-Sim protein-protein interaction domain, binds the C-terminal activation domain of myocardin and enhances myocardin-mediated transcriptional activation of VSMC-specific, CArG-containing promoters, including the VSMC-specific genes SM22 and myosin heavy chain. Suppression of endogenous SRC3 expression by specific small interfering RNA attenuates myocardin transcriptional activation in cultured cells. The SRC3-myocardin interaction identifies a site of convergence for nuclear hormone receptor-mediated and VSMC-specific gene regulation and suggests a possible mechanism for the vascular protective effects of estrogen on vascular injury.