Madin-Darby Canine Kidney II Cells Research Articles

PH-dependent pyridoxine transport by SLC19A2 and SLC19A3: Implications for absorption in acidic microclimates

SLC19A2 and SLC19A3, also known as thiamine transporters (THTR) 1 and 2, respectively, transport the positively charged thiamine (vitamin B1) into cells to enable its efficient utilization. SLC19A2 and SLC19A3 are also known to transport structurally unrelated cationic drugs, such as metformin, but whether this charge selectivity extends to other molecules, such as pyridoxine (vitamin B6), is unknown. We tested this possibility using Madin-Darby canine kidney II (MDCKII) cells and human embryonic kidney 293 (HEK293) cells for transfection experiments, and also using Caco-2 cells as human intestinal epithelial model cells. The stable expression of SLC19A2 and SLC19A3 in MDCKII cells (as well as their transient expression in HEK293 cells) led to a significant induction in pyridoxine uptake at pH 5.5 compared with control cells. The induced uptake was pH-dependent, favoring acidic conditions over neutral to basic conditions, and protonophore-sensitive. It was saturable as a function of pyridoxine concentration, with an apparent Km of 37.8 and 18.5 μm, for SLC19A2 and SLC19A3, respectively, and inhibited by the pyridoxine analogs pyridoxal and pyridoxamine as well as thiamine. We also found that silencing the endogenous SLC19A3, but not SLC19A2, of Caco-2 cells with gene-specific siRNAs lead to a significant reduction in carrier-mediated pyridoxine uptake. These results show that SLC19A2 and SLC19A3 are capable of recognizing/transporting pyridoxine, favoring acidic conditions for operation, and suggest a possible role for these transporters in pyridoxine transport mainly in tissues with an acidic environment like the small intestine, which has an acidic surface microclimate.

Role of Equilibrative Nucleobase Transporter 1/SLC43A3 as a Ganciclovir Transporter in the Induction of Cytotoxic Effect of Ganciclovir in a Suicide Gene Therapy with Herpes Simplex Virus Thymidine Kinase.

A suicide gene therapy using herpes simplex virus thymidine kinase (HSV-TK) with ganciclovir (GCV) has been under development as a tumor-targeted therapy; however, the mechanism of cellular GCV uptake, which is prerequisite in the therapy, has not been clarified. In an attempt to resolve this situation and gain information to optimize HSV-TK/GCV system for cancer therapy, we found that human equilibrative nucleobase transporter 1 (ENBT1) can transport GCV with a Michaelis constant of 2.75 mM in Madin-Darby canine kidney II (MDCKII) cells stably transfected with this transporter. In subsequent experiments using green fluorescent protein (GFP)-tagged ENBT1 (GFP-ENBT1) and HSV-TK, the uptake of GCV (30 μM), which was minimal in MDCKII cells and unchanged by their transfection with HSV-TK alone, was increased extensively by their transfection with GFP-ENBT1, together with HSV-TK. Accordingly, cytotoxicity, which was assessed by the WST-8 cell viability assay after the treatment of those cells with GCV (30 μM) for 72 hours, was induced in those transfected with GFP-ENBT1, together with HSV-TK but not in those transfected with HSV-TK alone. These results suggest that ENBT1 could facilitate GCV uptake and thereby enhance cytotoxicity in HSV-TK/GCV system. We also identified Helacyton gartleri (HeLa) and HepG2 as cancer cell lines that are rich with ENBT1 and A549, HCT-15 and MCF-7 as those poor with ENBT1. Accordingly, the HSV-TK/GCV system was effective in inducing cytotoxicity in the former but not in the latter. Thus, ENBT1 was found to be a GCV transporter that could enhance the performance of HSV-TK/GCV suicide gene therapy.

Structural determinants of peripheral O-arylcarbamate FAAH inhibitors render them dual substrates for Abcb1 and Abcg2 and restrict their access to the brain

The blood–brain barrier (BBB) is the main entry route for chemicals into the mammalian central nervous system (CNS). Two transmembrane transporters of the ATP-binding cassette (ABC) family – breast cancer resistance protein (ABCG2 in humans, Abcg2 in rodents) and P-glycoprotein (ABCB1 in humans, Abcb1 in rodents) – play a key role in mediating this process. Pharmacological and genetic evidence suggests that Abcg2 prevents CNS access to a group of highly potent and selective O-arylcarbamate fatty-acid amidohydrolase (FAAH) inhibitors, which include the compound URB937 (cyclohexylcarbamic acid 3′-carbamoyl-6-hydroxybiphenyl-3-yl ester). To define structure-activity relationships of the interaction of these molecules with Abcg2, in the present study we tested various peripherally restricted and non-restricted O-arylcarbamate FAAH inhibitors for their ability to serve as transport substrates in monolayer cultures of Madin-Darby Canine Kidney-II (MDCKII) cells over-expressing Abcg2. Surprisingly, we found that the majority of compounds tested – even those able to enter the CNS in vivo – were substrates for Abcg2 in vitro. Additional experiments in MDCKII cells overexpressing ABCB1 revealed that only those compounds that were dual substrates for ABCB1 and Abcg2 in vitro were also peripherally restricted in vivo. The extent of such restriction seems to depend upon other physicochemical features of the compounds, in particular the polar surface area. Consistent with these in vitro results, we found that URB937 readily enters the brain in dual knockout mice lacking both Abcg2 and Abcb1, whereas it is either partially or completely excluded from the brain of mice lacking either transporter alone. The results suggest that Abcg2 and Abcb1 act together to restrict the access of URB937 to the CNS.

In vitro transport characteristics of EFdA, a novel nucleoside reverse transcriptase inhibitor using Caco-2 and MDCKII cell monolayers

4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is a novel nucleoside reverse transcriptase inhibitor with a unique mechanism of action and highly potent activity against both wild-type and clinically relevant drug resistant HIV-1 variants. Furthermore, in vivo efficacy and safety evaluations have shown EFdA to be a promising therapeutic candidate for use in the treatment of HIV infection. However, little is known about the pharmacokinetic and biopharmaceutical properties of EFdA. In this study, we evaluated cellular EFdA transport using Caco-2 and Madin-Darby Canine Kidney II (MDCKII) in vitro cell models. Studies using Caco-2 cell monolayers showed that EFdA efflux ratios were >2.0, suggesting that active drug transport mechanisms may play a role in EFdA flux. ABCB1 transporter (PGP1) inhibition was assessed using the acetomethoxy derivate of calcein (calcein-AM) as a fluorescent probe in both wild-type MDCKII and PGP1 overexpressing MDCKII cells. Nonetheless, our data showed that EFdA is not a substrate of PGP1. Additionally, comparative bidirectional flux of EFdA and Lucifer yellow (LY, a well-known paracellular marker) was studied over a range of EFdA concentrations. In MDCKII monolayers, EFdA had an apparent permeability coefficient (Papp) (a–b) of <1×10−6cm/s. The Papp values significantly increased in the presence of the paracellular permeability enhancer, indicating that EFdA primarily permeates via the paracellular route.

Establishment of MDCKII Cell Monolayer with Metabolic Activity by CYP3A4 Transduced with Recombinant Adenovirus

This study aims to establish an in vitro system that can assess intestinal first-pass metabolism of CYP3A4 substrate drugs using adenoviral transduction. Madin-Darby canine kidney II (MDCKII) cells were used as a model of intestinal epithelial cells. Recombinant adenovirus expressing green fluorescent protein (AdGFP) and CYP3A4 (AdCYP3A4) was used as vectors. On day 2 after seeding MDCKII cells onto a semipermeable membrane, cells were infected with each adenovirus vector at various MOIs (multiplicities of infection) ranging from 0 to 200. On day 5, cell monolayers were used for drug transport study. The expression of GFP in monolayers of MDCKII cells transduced with AdGFP increased MOI-dependently and adenoviral infection showed no effect on the membrane permeability of drugs. The metabolite formation rate of midazolam, a CYP3A4 substrate, in the permeation process of a monolayer linearly increased with an increase in the MOI of AdCYP3A4. When the period that vectors and cells were located adjacent to each other was prolonged, the rate increased 2-fold compared with that calculated from a result with a monolayer obtained from a shorter period of adjacency. This study indicates that monolayers of MDCKII cells transduced with AdCYP3A4 have the potential to enable estimation of the first-pass metabolism by CYP3A4 in the intestinal absorption process.

PDZK1 Regulates Breast Cancer Resistance Protein in Small Intestine

Transporter adaptor protein PDZK1 regulates several influx transporters for xenobiotics and nutrients in small intestine, and their expression on the apical membrane is diminished in pdzk1 gene knockout [pdzk1(-/-)] mice. In the present study, we initially attempted to use pdzk1(-/-) mice to functionally identify influx transporters responsible for intestinal absorption of cimetidine. Contrary to our expectation, the plasma concentration of cimetidine after oral administration to pdzk1(-/-) mice was higher than that in wild-type mice, and the double peaks of plasma concentration found in wild-type mice were not observed in pdzk1(-/-) mice. Western blot analysis of intestinal brush-border membranes revealed that expression of breast cancer resistance protein (BCRP) but not of P-glycoprotein is reduced in pdzk1(-/-) mice. This result was compatible with the reduction of apical localization of BCRP in pdzk1(-/-) mice assessed by immunohistochemical analysis. Transcellular transport of cimetidine in the basal-to-apical direction in Madin-Darby canine kidney II (MDCKII) cells stably expressing both BCRP and PDZK1 (MDCKII/BCRP/PDZK1) was higher than that in MDCKII cells stably expressing BCRP (MDCKII/BCRP) cells. Moreover, MDCKII/BCRP/PDZK1 cells are more resistant than MDCKII/BCRP cells to the cytotoxicity of the anticancer agent 7-ethyl-10-hydroxycamptothecin (SN-38), which is a substrate of BCRP. These results were consistent with the higher expression of BCRP on apical membranes in MDCKII/BCRP/PDZK1 cells. Pull-down and immunoprecipitation studies revealed a physical interaction between BCRP and PDZK1. Taken together, these findings demonstrate that PDZK1 plays a pivotal role in the apical localization of BCRP. This is the first identification of a regulatory protein that physically interacts with and regulates BCRP in small intestine in vivo.

Enhancement of oral bioavailability of 20(S)-ginsenoside Rh2 through improved understanding of its absorption and efflux mechanisms.

The development of 20(S)-ginsenoside Rh2 (Rh2s) as a chemoprevention agent is limited by its low oral bioavailability. The goals of this study were to determine the mechanisms responsible for its poor oral absorption and to improve its bioavailability by overcoming the barrier to its absorption. Comprehensive studies were conducted using the following models: 1) monolayers of Caco-2, parental, and multidrug resistance gene (MDR1)-overexpressing Madin-Darby canine kidney II (MDCKII) cells; 2) pharmacokinetics in wild-type (WT) FVB, MDR1a/b knockout [MDR1a/b⁻/⁻] FVB, and A/J mice; and 3) intestinal perfusion in WT, MDR1a/b⁻/⁻ FVB, and A/J mice. Two P-glycoprotein (P-gp) inhibitors, verapamil and cyclosporine A, substantially decreased the efflux ratio of Rh2s from 28.5 to 1.0 and 1.2, respectively, in Caco-2 cells. The intracellular concentrations of Rh2s were also significantly increased (2.3- and 3.9-fold) in the presence of inhibitors. Similar results were obtained when transcellular transport of Rh2s were determined using MDR1-overexpressing MDCKII cells in the absence or presence of cyclosporine A. Compared with WT mice, the plasma C(max) and AUC₀-∞ of Rh2s were substantially increased by 17- and 23-fold in MDR1a/b⁻/⁻ FVB mice, respectively. In the A/J mice, the oral bioavailability of Rh2s (0.94% at 5 mg/kg and 0.52% at 20 mg/kg) was substantially increased by P-gp inhibitor to 33.18 and 27.14%, respectively. As expected, deletion or inhibition of P-gp significantly increased absorption and steady-state plasma concentration of Rh2s in a mouse intestinal perfusion model. In conclusion, Rh2s is a good substrate of P-gp, and inhibition of P-gp can significantly enhance its oral bioavailability.

Effects of naturally occurring G103D point mutation of AQP5 on its water permeability, trafficking and cellular localization in the submandibular gland of rats

AQPs (aquaporins) are water channel proteins that are expressed in almost all living things. In mammalians, 13 members of AQPs (AQP0-12) have been identified so far. AQP5 is known to be expressed mostly in the exocrine cells, including the salivary gland acinar cells. A naturally occurring point mutation (G308A, Gly103 > Asp103) was earlier found in the rat AQP5 gene [Murdiastuti, Purwanti, Karabasil, Li, Yao, Akamatsu, Kanamori and Hosoi (2006) Am. J. Physiol. 291, G1081-G1088]; in this mutant, the rate of initial saliva secretion under stimulated and unstimulated conditions is less than that for the wt (wild-type) animals. Here the mutant molecule was characterized in detail. Using the Xenopus oocyte system, we demonstrated the mutant AQP5 to have water permeability almost the same as that of the wt molecule. Mutant and wt AQP5s, tagged with GFP (green fluorescent protein; GFP-AQP5s) and expressed in polarized MDCK-II (Madin-Darby canine kidney II) cells, first appeared in the vesicular structure(s) in the cytoplasm, and were translocated to the upper plasma membrane or apical membrane during cultivation, with the mutant GFP-AQP5 being translocated less efficiently. Thapsigargin and H-89 both induced translocation in vitro of either molecule, whereas colchicine inhibited this activity; the fraction of cells showing apical localization of mutant GFP-AQP5 was less than that showing that of the wt molecule under any of the experimental conditions used. In the mutant SMG (submandibular gland) tissue, localization of AQP5 in the apical membrane of acinar cells was extremely reduced. Vesicular structures positive for AQP5 and present in the cytoplasm of the acinar cells were co-localized with LAMP2 (lysosome-associated membrane protein 2) or cathepsin D in the mutant gland, whereas such co-localizations were very rare in the wt gland, suggesting that the mutant molecules largely entered lysosomes for degradation. Replacement of highly conserved hydrophobic Gly103 with strongly hydrophilic Asp103 in rat AQP5, though it did not affect water permeability, may possibly have resulted in less efficient membrane trafficking and increased lysosomal degradation, leading to its lower expression in the apical membrane of the acinar cells in the SMG.

Trafficking of GFP-AQP5 chimeric proteins conferred with unphosphorylated amino acids at their PKA-target motif (152SRRTS) in MDCK-II cells

Three constructs having mutated PKA-target motif at (152)SRRTS of AQP5, an exocrine type water channel, were prepared and fused to C-terminus of green fluorescence protein cDNA to examine the effects of blocking of phosphorylation at (152)SRRTS (a consensus PKA-target motif of AQP5) on translocation or trafficking of the chimeric proteins expressed in the Madin-Darby canine kidney-II (MDCK-II) cells. H-89 treatment increased translocation of wild-type GFP-AQP5 to the apical membrane. All 3 mutant molecules translocated 1.5 to 2 times more than the control wild-type GFP-AQP5. Colchicine but not cytochalasin B inhibited the translocation of wild-type GFP-AQP5. Present results suggest dephosphorylation of this consensus sequence increase GFP-AQP5 translocation, and that microtubules but not microfilaments are involved in this event.

A novel role of the C-terminus of b0,+AT in the ER–Golgi trafficking of the rBAT–b0,+AT heterodimeric amino acid transporter

The heterodimeric complex composed of rBAT (related to b(0,+) amino acid transporter), a single-membrane-spanning glycosylated heavy chain, and b(0,+)AT, a putative 12-membrane-spanning non-glycosylated light chain, is an amino acid transporter that mediates the activity of system b(0,+), a major apical transport system for cystine and dibasic amino acids in renal proximal tubule and small intestine. The C-terminus of b(0,+)AT has been proposed to play an important role in the functional expression of the heterodimeric transporters. In the present study, to reveal the roles of the C-terminus, we analysed b(0,+)AT mutants whose C-termini were sequentially deleted or replaced by site-directed mutagenesis in polarized MDCKII (Madin-Darby canine kidney II), non-polarized HEK-293 (human embryonic kidney-293) and HeLa cells. Although the deletion of C-terminus of b(0,+)AT did not affect the formation of a heterodimer with rBAT, it resulted in the loss of apparent transport function, owing to the failure of the plasma-membrane targeting of rBAT-b(0,+)AT heterodimeric complex associated with incomplete glycosylation of rBAT. A motif-like sequence Val(480)-Pro(481)-Pro(482) was identified in the C-terminus of b(0,+)AT to be responsible for the C-terminus action in promoting the trafficking of rBAT-b(0,+)AT heterodimeric complex from the ER (endoplasmic reticulum) to Golgi apparatus. This is, to our knowledge, the first demonstration of the active contribution of the C-terminus of a light-chain subunit to the intracellular trafficking of heterodimeric transporters. Because the motif-like sequence Val(480)-Pro(481)-Pro(482) is well conserved among the C-termini of light-chain subunits, common regulatory mechanisms could be proposed among heterodimeric amino acid transporters.

Development and validation of an assay for the quantitative determination of cladribine nucleotides in MDCKII cells and culture medium using weak anion‐exchange liquid chromatography coupled with tandem mass spectrometry

The development and validation of an assay for the quantitative analysis of cladribine mono-, di- and triphosphate (2-chloro, 2'-deoxyadenosine 5'-mono-, di- and triphosphate or 2CdAMP, 2CdADP and 2CdATP) in culture medium (Optimem) and cell lysate is described. Cladribine mono- and diphosphate reference compounds were obtained by thermal degradation of cladribine triphosphate. The reference compounds were characterized using ion-pairing reversed-phase high-performance liquid chromatography with ultraviolet detection. The bioanalytical assay for 2CdAMP, 2CdADP and 2CdATP is based on weak anion-exchange liquid chromatography coupled with tandem mass spectrometry in the positive ion mode (WAXLC/MS/MS). A fused-silica electrospray capillary was used instead of a stainless steel electrospray capillary to minimize adsorption of analytes and thus decrease variation in the analyte signals. Dynamic ranges of 1.11-27.7, 0.550-55.0 and 1.31-52.3 nM for 2CdAMP, 2CdADP and 2CdATP, respectively, were validated in culture medium and cell lysate. Optimem samples required stabilization with 30% methanol to prevent conversion of 2CdATP into 2CdAMP and 2CdADP. All intra- and interday accuracies and precisions were within +/-20%. The stability of the compounds was assessed under various analytically relevant conditions. The method was successfully used to investigate cladribine nucleotide transport in vitro in Madin-Darby canine kidney II (MDCKII) cells.

PATJ regulates directional migration of mammalian epithelial cells

Directional migration is important in wound healing by epithelial cells. Recent studies have shown that polarity proteins such as mammalian Partitioning-defective 6 (Par6), atypical protein kinase C (aPKC) and mammalian Discs large 1 (Dlg1) are crucial not only for epithelial apico-basal polarity, but also for directional movement. Here, we show that the protein associated with Lin seven 1 (PALS1)-associated tight junction protein (PATJ), another evolutionarily conserved polarity protein, is also required for directional migration by using a wound-induced migration assay. In addition, we found that aPKC and Par3 localize to the leading edge during migration of epithelia and that PATJ regulates their localization. Furthermore, our results show that microtubule-organizing centre orientation is disrupted in PATJ RNA interference (RNAi) MDCKII (Madin-Darby canine kidney II) cells during migration. Together, our data indicate that PATJ controls directional migration by regulating the localization of aPKC and Par3 to the leading edge. The migration defect in PATJ RNAi cells seems to be due to the disorganization of the microtubule network induced by mislocalization of polarity proteins.

The caspase-3 cleavage product of the plasma membrane Ca2+-ATPase 4b is activated and appropriately targeted

The calmodulin-activated transporter hPMCA4 (human plasma membrane Ca2+-ATPase isoform 4) is a target for cleavage by caspase-3 during apoptosis. We have demonstrated that caspase-3 generates a 120 kDa fragment of this pump which lacks the complete autoinhibitory sequence [Paszty, Verma, Padanyi, Filoteo, Penniston and Enyedi (2002) J. Biol. Chem. 277, 6822-6829]. In the present study we analysed further the characteristics of the fragment of hPMCA4b produced by caspase-3. We did this by overexpressing the caspase-3 cleavage product of hPMCA4b in COS-7 and MDCKII (Madin-Darby canine kidney II) cells. This technique made it possible to clearly define the properties of this fragment, and we showed that it is constitutively active, as it forms a phosphoenzyme intermediate and has high Ca2+ transport activity in the absence of calmodulin. When this fragment of hPMCA4b was stably expressed in MDCKII cell clones, it was targeted without degradation to the basolateral plasma membrane. In summary, our studies emphasize that the caspase-3 cleavage product of hPMCA4b is constitutively active, and that the C-terminus is not required for proper targeting of hPMCA4b to the plasma membrane. Also, for the first time, we have generated cell clones that stably express a constitutively active PMCA.

Internalization of calcium oxalate crystals by renal tubular cells: A nephron segment–specific process?

Crystal retention in the kidney is caused by the interaction between crystals and the cells lining the renal tubules. These interactions involve crystal attachment, followed by internalization or not. Here, we studied the ability of various renal tubular cell lines to internalize calcium oxalate monohydrate (COM) crystals. Crystal-cell interactions are studied by light-, electron-, and confocal microscopy with cells resembling the renal proximal tubule [porcine kidney (LLC-PK1)], proximal/distal tubule [Madin-Darby canine kidney II (MDCK-II)], and distal tubule and/or collecting ducts [(Madin-Darby canine kidney I (MDCK-I), rat cortical collecting duct 1 (RCCD1)]. Crystal-binding strength and internalization are characterized and quantified with radiolabeled COM. Microscopy studies showed that crystals were firmly embedded in the membranes of LLC-PK1 and MDCK-II cells to be subsequently internalized. On the other hand, crystals bound only loosely to MDCK-I and RCCD1 and were not taken up by these cells. Crystal uptake by LLC-PK1 and MDCK-II, expressed in microg/10(6) cells, is temperature-dependent and gradually increases from 0.88 and 0.15 in 30 minutes, respectively, to 4.70 and 3.85, respectively, after five hours, whereas these values never exceeded background levels in MDCK-I and RCCD1 cells. The adherence of COM crystals to renal cells with properties of the proximal tubule is inevitable and actively followed by their uptake, whereas crystals attached to cells resembling the distal tubule and/or collecting duct are not internalized. Since crystal formation usually occurs in segments beyond the renal proximal tubule, crystal uptake may be of less importance in the etiology of idiopathic calcium oxalate stone disease.

Agonist-regulated Interaction between α2-Adrenergic Receptors and Spinophilin

Previously, we demonstrated that the third intracellular (3i) loop of the heptahelical alpha2A-adrenergic receptor (alpha2A AR) is critical for retention at the basolateral surface of polarized Madin-Darby canine kidney II (MDCKII) cells following their direct targeting to this surface. Findings that the 3i loops of the D2 dopamine receptors interact with spinophilin (Smith, F. D., Oxford, G. S., and Milgram, S. L. (1999) J. Biol. Chem. 274, 19894-19900) and that spinophilin is enriched beneath the basolateral surface of polarized MDCK cells prompted us to assess whether alpha(2)AR subtypes might also interact with spinophilin. [35S]Met-labeled 3i loops of the alpha2A AR (Val(217)-Ala(377)), alpha2BAR (Lys(210)-Trp(354)), and alpha2CAR (Arg(248)-Val(363)) subtypes interacted with glutathione S-transferase-spinophilin fusion proteins. These interactions could be refined to spinophilin amino acid residues 169-255, in a region between spinophilin's F-actin binding and phosphatase 1 regulatory domains. Furthermore, these interactions occur in intact cells in an agonist-regulated fashion, because alpha2A AR and spinophilin coimmunoprecipitation from cells is enhanced by prior treatment with agonist. These findings suggest that spinophilin may contribute not only to alpha2 AR localization but also to agonist modulation of alpha2AR signaling.

Identification of a Novel β-Catenin-Interacting Protein

Cadherin is a well-known cell–cell adhesion molecule, and it binds to β-catenin, which in turn binds to α-catenin. However, little is known about the regulatory mechanism underlying the cadherin-mediated cell–cell adhesion. Here we purified two novel β-catenin-interacting proteins with molecular masses of 180 kDa (p180) and 150 kDa (p150) from bovine brain cytosol by using glutathione S-transferase (GST)–β-catenin affinity column chromatography. Mass spectral analysis revealed p180 to be identical to KIAA0313 which has a putative Rap1 guanine nucleotide exchange factor (GEF) domain and p150 to be the same as KIAA0705 which has a high degree of sequence similarity to the synaptic scaffolding molecule (S-SCAM), which binds β-catenin and KIAA0313 in the yeast two-hybrid system and overlay assay, respectively (Ide et al., Biochem. Biophys. Res. Commun. 256, 456–461, 1999; Ohtsuka et al., Biochem. Biophys. Res. Commun. 265, 38–44, 1999). β-Catenin was coimmunoprecipitated with KIAA0313 in Madin–Darby canine kidney II (MDCKII) cells, bovine brain cytosol, and EL cells. KIAA0313 and β-catenin were partly colocalized at sites of cell–cell contact in MDCKII cells. Taken together, our data suggest that KIAA0313 associates with β-catenin through KIAA0705 in vivo at sites of cell–cell contact.

Polarity of Constitutive and Regulated von Willebrand Factor Secretion by Transfected MDCK-II Cells

Von Willebrand factor (vWF), synthesized by endothelial cells, is both rapidly secreted by the constitutive pathway and stored in Weibel–Palade bodies. Secretion from these organelles occurs upon activation of the protein kinase C signal transduction pathway and yields highly multimerized vWF. Highly multimerized vWF acts as a more effective adhesive ligand than the lower molecular weight forms that are constitutively secreted. We employed the extensively characterized polar Madin–Darby Canine Kidney II (MDCK-II) epithelial cell line, stably transfected with full-length vWF cDNA or deletion mutants thereof, to gain insight in the polarity of vWF secretion by either one of the two pathways. Immunofluorescence analysis and metabolic labeling experiments revealed that multimeric “wild-type” vWF is stored in MDCK-II cells and released upon stimulation with phorbol esters. Furthermore, we show that 62.0 ± 3.8% of constitutively secreted and 83.2 ± 6.6% of the regulated secreted wild-type vWF is encountered at the apical side of the cell. The polarity of the constitutive secretion of deletion mutant vWFdelD′D3 is similar to that of constitutively secreted wild-type vWF, whereas deletion mutant vWFdelD1D2 displays no polar secretion (50.1 ± 5.7% apical).