The mixing of gastric and pancreatic juice subjects the jejunum to unique ionic conditions with high luminal CO₂ tension and HCO₃ ⁻ concentration. We investigated the role of the small intestinal apical anion exchangers PAT-1 (Slc26a6) and DRA (Slc26a3) in basal and CO₂/HCO₃ ⁻-stimulated jejunal fluid absorption. Single pass perfusion of jejunal segments was performed in anaesthetised wild type (WT) as well as in mice deficient in DRA, PAT-1, Na⁺/H⁺ exchanger 3 (NHE3) or NHE2, and in carbonic anhydrase II (CAII). Unbuffered saline (pH 7.4) perfusion of WT jejunum resulted in fluid absorption and acidification of the effluent. DRA-deficient jejunum absorbed less fluid than WT, and acidified the effluent more strongly, consistent with its action as a Cl⁻/HCO₃ ⁻ exchanger. PAT-1-deficient jejunum also absorbed less fluid but resulted in less effluent acidification. Switching the luminal solution to a 5 % CO₂/HCO₃ ⁻ buffered solution (pH 7.4), resulted in a decrease in jejunal enterocyte pH_i in all genotypes, an increase in luminal surface pH and a strong increase in fluid absorption in a PAT-1- and NHE3- but not DRA-, CAII, or NHE2-dependent fashion. Even in the absence of luminal Cl⁻, luminal CO₂/HCO₃ ⁻ augmented fluid absorption in WT, CAII, NHE2- or DRA-deficient, but not in PAT-1- or NHE3-deficient mice, indicating the likelihood that PAT-1 serves to import HCO₃ ⁻ and NHE3 serves to import Na⁺ under these circumstances. The results suggest that PAT-1 plays an important role in jejunal Na⁺HCO₃ ^– reabsorption, while DRA absorbs Cl⁻ and exports HCO₃ ⁻ in a partly CAII-dependent fashion. Both PAT-1 and DRA significantly contribute to intestinal fluid absorption and enterocyte acid/base balance but are activated by different ion gradients.

Objective— To examine the contribution of the scavenger receptor (SR) BI to the mechanism by which high-density lipoprotein (HDL) acquires paraoxonase-1 (PON1). Methods and Results— Serum PON1 activity contributes to the antioxidant capacity of HDLs and is suggested to be an independent risk factor for atherosclerosis. The association of PON1 with HDL is a major determinant of its serum activity levels. PON1 secretion was studied in stably transfected Chinese hamster ovary and HepG2 models. Complementary analyses were performed in transgenic models. Modulation of SR-BI expression, by SR-BI small and interfering RNA knockdown and pharmacologically, correlated with significant changes ( P <0.01) in PON1 secretion to HDLs and very-low-density lipoproteins. Block lipid transport-1 (BLT1), which increases the affinity of HDL for SR-BI without modulating its expression, was associated with significant increases in secretion. Downregulating postsynaptic density 95/disc-large/zona occludens kinase in HepG2 reduced cell SR-BI protein and lowered enzyme secretion. Serum PON1 activity was significantly reduced in postsynaptic density 95/disc-large/zona occludens kinase knockout mice. Conclusion— The present study identifies SR-BI as a major determinant of the capacity of HDL to acquire PON1. It reinforces the concept of the receptor as a docking molecule, allowing communication between HDL and the cell, and extends the importance of SR-BI to HDL metabolism and function.

The actin-regulated transcription factor MRTF-A represents a central relay in mechanotransduction and controls a subset of SRF-dependent target genes. However, gain-of-function studies in vivo are lacking. Here we characterize a conditional MRTF-A transgenic mouse model. While MRTF-A gain-of-function impaired embryonic development, induced expression of constitutively active MRTF-A provoked rapid hepatocyte ballooning and liver failure in adult mice. Specific expression in the intestinal epithelium caused an erosive architectural distortion, villus blunting, cryptal hyperplasia and colonic inflammation, resulting in transient weight loss. Organoids from transgenic mice repeatedly induced in vitro showed impaired self-renewal and defective cryptal compartments. Mechanistically, MRTF-A gain-of-function decreased proliferation and increased apoptosis, but did not induce fibrosis. MRTF-A targets including Acta2 and Pai-1 were induced, whereas markers of stem cells and differentiated cells were reduced. Our results suggest that activated MRTF-A in the intestinal epithelium shifts the balance between proliferation, differentiation and apoptosis.

In their GTP-bound (active) form, Rab proteins interact with effector proteins that control downstream signaling. One such Rab15 effector is Rep15, which is known to have a role in receptor recycling from the endocytic recycling compartment but otherwise remains poorly characterized. Here, we report the characterization of the Rep15:Rab15 interaction and identification of Rab3 paralogs and Rab34 as Rep15 interacting partners from a yeast two-hybrid assay. Biochemical validation of the interactions is presented and crystal structures of the Rep15:Rab3B and Rep15:Rab3C complexes provide additional mechanistic insight. We find that Rep15 adopts a globular structure that is distinct from other reported Rab15, Rab3 and Rab34 effectors. Structure-based mutagenesis experiments explain the Rep15:Rab interaction specificity. Rep15 depletion in U138MG glioblastoma cells impairs cell proliferation, cell migration and receptor recycling, underscoring the need for further clarification of the role of Rep15 in cancer.

Myoblast fusion is essential for the formation, growth, and regeneration of skeletal muscle, but the molecular mechanisms that govern fusion and myofiber formation remain poorly understood. Past studies have shown an important role of the actin cytoskeleton and actin regulators in myoblast fusion. The Cyclase-Associated Proteins (CAP) 1 and 2 recently emerged as critical regulators of actin treadmilling in higher eukaryotes including mammals. Whilst the role of CAP2 in skeletal muscle development and function is well characterized, involvement of CAP1 in this process remains elusive. Here we report that CAP1, plays a critical role in cytoskeletal remodeling during myoblast fusion and formation of myotubes. Cap1 mRNA and protein are expressed in both murine C2C12 and human LHCN-M2 myoblasts, but their abundance decreases during myogenic differentiation. Perturbing the temporally controlled expression of CAP1 by overexpression or CRISPR-Cas9 mediated knockout impaired actin rearrangement, myoblast alignment, expression of profusion molecules, differentiation into multinucleated myotubes, and myosin heavy chain expression. Endogenous Cap1 expression is post-transcriptionally downregulated during differentiation by canonical myomiRs miR-1, miR-133, and miR-206, which have conserved binding sites at the 3′ UTR of the Cap1 mRNA. Deletion of the endogenous 3′ UTR by CRISPR-Cas9 in C2C12 cells phenocopies overexpression of CAP1 by inhibiting myotube formation. Our findings implicates Cap1 and its myomiR-mediated downregulation in the myoblast fusion process and the generation of skeletal muscle.

Skeletal muscle differentiation requires coordinated cytoskeletal remodeling and transcriptional reprogramming. Members of the actin-depolymerizing factor/Cofilin family regulate actin filament turnover, yet the isoform-specific functions of Cofilin-1 and Cofilin-2 during myogenesis remain incompletely understood. Murine C2C12 myoblasts were used to investigate the expression and function of Cofilin isoforms during differentiation. CRISPR/Cas9-mediated knockout, shRNA-mediated knockdown, immunofluorescence, gene expression analyses, MRTF reporter assays, and pharmacological inhibition of LIM kinase were used to assess myogenic progression and signaling. Myogenic differentiation was accompanied by a pronounced isoform transition characterized by progressive downregulation of Cofilin-1 and Actin-Depolymerizing Factor together with increased expression of Cofilin-2. Loss of Cofilin-1 resulted in marked morphological abnormalities, impaired cell-cycle exit, elevated MRTF activity, and reduced myotube formation, whereas Cofilin-2 deficiency produced comparatively mild effects during early differentiation. Importantly, partial reduction of Cofilin-1 enhanced myoblast fusion, while stronger depletion impaired differentiation, demonstrating a dosage-sensitive requirement for Cofilin-1. In addition, Cofilin-1 activity was dynamically regulated by transient LIM kinase-mediated phosphorylation during early differentiation, and inhibition of this pathway disrupted myogenic progression. Analyses of mRNA and protein stability indicated that isoform switching is primarily regulated at the level of gene expression. Cofilin-1 functions as a dosage-sensitive regulator of myogenic progression that links cytoskeletal remodeling to signaling pathways controlling proliferation and differentiation. Coordinated regulation of Cofilin-1 expression and activity, together with the transition to Cofilin-2, is required for efficient myoblast fusion and muscle formation.