Identification of intestinal ion transport defects in microvillus inclusion disease.

Loss of function mutations in the actin motor myosin Vb (Myo5b) lead to microvillus inclusion disease (MVID) and death in newborns and children. MVID results in secretory diarrhea, brush border (BB) defects, villus atrophy, and microvillus inclusions (MVIs) in enterocytes. How loss of Myo5b results in increased stool loss of chloride (Cl(-)) and sodium (Na(+)) is unknown. The present study used Myo5b loss-of-function human MVID intestine, polarized intestinal cell models of secretory crypt (T84) and villus resembling (CaCo2BBe, C2BBe) enterocytes lacking Myo5b in conjunction with immunofluorescence confocal stimulated emission depletion (gSTED) imaging, immunohistochemical staining, transmission electron microscopy, shRNA silencing, immunoblots, and electrophysiological approaches to examine the distribution, expression, and function of the major BB ion transporters NHE3 (Na(+)), CFTR (Cl(-)), and SLC26A3 (DRA) (Cl(-)/HCO3 (-)) that control intestinal fluid transport. We hypothesized that enterocyte maturation defects lead villus atrophy with immature secretory cryptlike enterocytes in the MVID epithelium. We investigated the role of Myo5b in enterocyte maturation. NHE3 and DRA localization and function were markedly reduced on the BB membrane of human MVID enterocytes and Myo5bKD C2BBe cells, while CFTR localization was preserved. Forskolin-stimulated CFTR ion transport in Myo5bKD T84 cells resembled that of control. Loss of Myo5b led to YAP1 nuclear retention, retarded enterocyte maturation, and a cryptlike phenotype. We conclude that preservation of functional CFTR in immature enterocytes, reduced functional expression of NHE3, and DRA contribute to Cl(-) and Na(+) stool loss in MVID diarrhea.

Restoration of cytoskeletal and membrane tethering defects but not defects in membrane trafficking in the intestinal brush border of mice lacking both myosin Ia and myosin VI.

Myosin Ia (Myo1a), the most prominent plus-end directed motor and myosin VI (Myo6) the sole minus-end directed motor, together exert opposing tension between the microvillar (MV) actin core and the apical brush border (BB) membrane of the intestinal epithelial cell (IEC). Mice lacking Myo1a or Myo6 each exhibit a variety of defects in the tethering of the BB membrane to the actin cytoskeleton. Double mutant (DM) mice lacking both myosins revealed that all the defects observed in either the Myo1a KO or Snell's waltzer (sv/sv) Myo6 mutant mouse are absent. In isolated DM BBs, Myo1a crosslinks between MV membrane and MV actin core are absent but the gap (which is lost in Myo1a KO) between the MV core and membrane is maintained. Several myosins including Myo1c, d, and e and Myo5a are ectopically recruited to the BB. Consistent with the restoration of membrane tethering defects by one or more of these myosins, upward ATP-driven shedding of the BB membrane, which is blocked in the Myo1a KO, is restored in the DM BB. However, Myo1a or Myo6 dependent defects in expression of membrane proteins that traffic between the BB membrane and endosome (NaPi2b, NHE3, CFTR) are not restored. Compared to controls, Myo1a KO, sv/sv mice exhibit moderate and DM high levels of hypersensitivity to dextran sulfate sodium-induced colitis. Consistent with Myo1a and Myo6 playing critical roles in maintaining IEC integrity and response to injury, DM IECs exhibit increased numbers of apoptotic nuclei, above that reported for Myo1a KO.

Myosin 5b loss of function leads to defects in polarized signaling: implication for microvillus inclusion disease pathogenesis and treatment.

Microvillus inclusion disease (MVID) is an autosomal recessive condition resulting in intractable secretory diarrhea in newborns due to loss-of-function mutations in myosin Vb (Myo5b). Previous work suggested that the apical recycling endosomal (ARE) compartment is the primary location for phosphoinositide-dependent protein kinase 1 (PDK1) signaling. Because the ARE is disrupted in MVID, we tested the hypothesis that polarized signaling is affected by Myo5b dysfunction. Subcellular distribution of PDK1 was analyzed in human enterocytes from MVID/control patients by immunocytochemistry. Using Myo5b knockdown (kd) in Caco-2BBe cells, we studied phosphorylated kinases downstream of PDK1, electrophysiological parameters, and net water flux. PDK1 was aberrantly localized in human MVID enterocytes and Myo5b-deficient Caco-2BBe cells. Two PDK1 target kinases were differentially affected: phosphorylated atypical protein kinase C (aPKC) increased fivefold and phosohoprotein kinase B slightly decreased compared with control. PDK1 redistributed to a soluble (cytosolic) fraction and copurified with basolateral endosomes in Myo5b kd. Myo5b kd cells showed a decrease in net water absorption that could be reverted with PDK1 inhibitors. We conclude that, in addition to altered apical expression of ion transporters, depolarization of PDK1 in MVID enterocytes may lead to aberrant activation of downstream kinases such as aPKC. The findings in this work suggest that PDK1-dependent signaling may provide a therapeutic target for treating MVID.

Molecular motors and apical CFTR traffic in epithelia.

Intracellular protein traffic plays an important role in the regulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channels. Microtubule and actin-based motor proteins direct CFTR movement along trafficking pathways. As shown for other regulatory proteins such as adaptors, the involvement of protein motors in CFTR traffic is cell-type specific. Understanding motor specificity provides insight into the biology of the channel and opens opportunity for discovery of organ-specific drug targets for treating CFTR-mediated diseases.

Myosin Ia is required for CFTR brush border membrane trafficking and ion transport in the mouse small intestine.

In enterocytes of the small intestine, endocytic trafficking of CFTR channels from the brush border membrane (BBM) to the subapical endosomes requires the minus-end motor, myosin VI (Myo6). The subapical localization of Myo6 is dependent on myosin Ia (Myo1a) the major plus-end motor associated with the BBM, suggestive of functional synergy between these two motors. In villus enterocytes of the Myo1a KO mouse small intestine, CFTR accumulated in syntaxin-3 positive subapical endosomes, redistributed to the basolateral domain and was absent from the BBM. In colon, where villi are absent and Myo1a expression is low, CFTR exhibited normal localization to the BBM in the Myo1a KO similar to WT. cAMP-stimulated CFTR anion transport in the small intestine was reduced by 58% in the KO, while anion transport in the colon was comparable to WT. Co-immunoprecipitation confirmed the association of CFTR with Myo1a. These data indicate that Myo1a is an important regulator of CFTR traffic and anion transport in the BBM of villus enterocytes and suggest that Myo1a may power apical CFTR movement into the BBM from subapical endosomes. Alternatively, it may anchor CFTR channels in the BBM of villus enterocytes as was proposed for Myo1a's role in BBM localization of sucrase-isomaltase.

Sucrose octasulfate selectively accelerates thrombin inactivation by heparin cofactor II.

Inactivation of thrombin (T) by the serpins heparin cofactor II (HCII) and antithrombin (AT) is accelerated by a heparin template between the serpin and thrombin exosite II. Unlike AT, HCII also uses an allosteric interaction of its NH(2)-terminal segment with exosite I. Sucrose octasulfate (SOS) accelerated thrombin inactivation by HCII but not AT by 2000-fold. SOS bound to two sites on thrombin, with dissociation constants (K(D)) of 10 +/- 4 microm and 400 +/- 300 microm that were not kinetically resolvable, as evidenced by single hyperbolic SOS concentration dependences of the inactivation rate (k(obs)). SOS bound HCII with K(D) 1.45 +/- 0.30 mm, and this binding was tightened in the T.SOS.HCII complex, characterized by K(complex) of approximately 0.20 microm. Inactivation data were incompatible with a model solely depending on HCII.SOS but fit an equilibrium linkage model employing T.SOS binding in the pathway to higher order complex formation. Hirudin-(54-65)(SO(3)(-)) caused a hyperbolic decrease of the inactivation rates, suggesting partial competitive binding of hirudin-(54-65)(SO(3)(-)) and HCII to exosite I. Meizothrombin(des-fragment 1), binding SOS with K(D) = 1600 +/- 300 microm, and thrombin were inactivated at comparable rates, and an exosite II aptamer had no effect on the inactivation, suggesting limited exosite II involvement. SOS accelerated inactivation of meizothrombin 1000-fold, reflecting the contribution of direct exosite I interaction with HCII. Thrombin generation in plasma was suppressed by SOS, both in HCII-dependent and -independent processes. The ex vivo HCII-dependent process may utilize the proposed model and suggests a potential for oversulfated disaccharides in controlling HCII-regulated thrombin generation.

Factor XI contributes to thrombin generation in the absence of factor XII.

During surface-initiated blood coagulation in vitro, activated factor XII (fXIIa) converts factor XI (fXI) to fXIa. Whereas fXI deficiency is associated with a hemorrhagic disorder, factor XII deficiency is not, suggesting that fXI can be activated by other mechanisms in vivo. Thrombin activates fXI, and several studies suggest that fXI promotes coagulation independent of fXII. However, a recent study failed to find evidence for fXII-independent activation of fXI in plasma. Using plasma in which fXII is either inhibited or absent, we show that fXI contributes to plasma thrombin generation when coagulation is initiated with low concentrations of tissue factor, factor Xa, or alpha-thrombin. The results could not be accounted for by fXIa contamination of the plasma systems. Replacing fXI with recombinant fXI that activates factor IX poorly, or fXI that is activated poorly by thrombin, reduced thrombin generation. An antibody that blocks fXIa activation of factor IX reduced thrombin generation; however, an antibody that specifically interferes with fXI activation by fXIIa did not. The results support a model in which fXI is activated by thrombin or another protease generated early in coagulation, with the resulting fXIa contributing to sustained thrombin generation through activation of factor IX.

A classification system for cross-reactive material-negative factor XI deficiency.

The bleeding disorder associated with factor XI (fXI) deficiency is typically inherited as an autosomal recessive trait. However, some fXI mutations may be associated with dominant disease transmission. FXI is a homodimer, a feature that could allow certain mutations to exert a dominant-negative effect on wild-type fXI secretion through heterodimer formation. We describe 2 novel fXI mutations (Ser225Phe and Cys398Tyr) that form intracellular dimers, are secreted poorly, and exhibit dominant-negative effects on wild-type fXI secretion in cotransfection experiments. Available data now suggest that mutations associated with crossreactive material-negative fXI deficiency fall into 1 of 3 mechanistic categories: (1) mutations that reduce or prevent polypeptide synthesis, (2) polypeptides that fail to form intracellular dimers and are retained in cells as monomers, and (3) polypeptides that form dimers that are not secreted. The latter category likely accounts for many cases of dominant disease transmission.

Dominant factor XI deficiency caused by mutations in the factor XI catalytic domain.

The bleeding diathesis associated with hereditary factor XI (fXI) deficiency is prevalent in Ashkenazi Jews, in whom the disorder appears to be an autosomal recessive condition. The homodimeric structure of fXI implies that the product of a single mutant allele could confer disease in a dominant manner through formation of heterodimers with wild-type polypeptide. We studied 2 unrelated patients with fXI levels less than 20% of normal and family histories indicating dominant disease transmission. Both are heterozygous for single amino acid substitutions in the fXI catalytic domain (Gly400Val and Trp569Ser). Neither mutant is secreted by transfected fibroblasts. In cotransfection experiments with a wild-type fXI construct, constructs with mutations common in Ashkenazi Jews (Glu117Stop and Phe283Leu) and a variant with a severe defect in dimer formation (fXI-Gly350Glu) have little effect on wild-type fXI secretion. In contrast, cotransfection with fXI-Gly400Val or fXI-Trp569Ser reduces wild-type secretion about 50%, consistent with a dominant negative effect. Immunoprecipitation of cell lysates confirmed that fXI-Gly400Val forms intracellular dimers. The data support a model in which nonsecretable mutant fXI polypeptides trap wild-type polypeptides within cells through heterodimer formation, resulting in lower plasma fXI levels than in heterozygotes for mutations that cause autosomal recessive fXI deficiency.

Cloning and characterization of the human factor XI gene promoter: transcription factor hepatocyte nuclear factor 4alpha (HNF-4alpha ) is required for hepatocyte-specific expression of factor XI.

Factor XI is the zymogen of a plasma protease produced primarily in liver that is required for normal blood coagulation. We cloned approximately 2600 base pairs of the human factor XI gene upstream of exon one, identified transcription start sites, and conducted a functional analysis. Luciferase reporter assays demonstrate that the 381 base pairs upstream of exon one are sufficient for maximum promoter activity in HepG2 hepatocellular carcinoma cells. The removal of 19 base pairs between -381 and -363 results in a nearly complete loss of promoter activity. This region contains the sequence ACTTTG, a motif required for binding of the transcription factor hepatocyte nuclear factor 4alpha (HNF-4alpha) to the promoters of several genes. Gel mobility shift assays using HepG2 or rat hepatocyte nuclear extract confirm HNF-4alpha binds between bp -375 and -360. Scrambling the ACTTTG motif completely abolishes promoter activity in luciferase assays. The factor XI promoter functions poorly when transfected into HeLa carcinoma cells, and gel mobility shift experiments with HeLa nuclear extracts demonstrate no HNF-4alpha binding to the ACTTTG sequence. When a rat HNF-4alpha expression construct is co-transfected into HeLa cells, factor XI promoter activity is enhanced approximately 10-fold. We conclude that HNF-4alpha is required for hepatocyte-specific expression of factor XI.