Differences in the apical and basolateral pathways for glycosaminoglycan biosynthesis in Madin-Darby canine kidney cells

Serglycin with a green fluorescent protein tag (SG-GFP) expressed in epithelial Madin-Darby canine kidney cells is secreted mainly (85%) into the apical medium, but the glycosaminoglycan (GAG) chains on the SG-GFP protein core secreted basolaterally (15%) carry most of the sulfate added during biosynthesis (Tveit et al. (2005) J. Biol. Chem., 280, 29596-29603). Here we report further differences in apical and basolateral GAG synthesis. The less intensely sulfated chondroitin sulfate (CS) chains on apically secreted SG-GFP are longer than CS chains attached to basolateral SG-GFP, whereas the heparan sulfate (HS) chains are of similar lengths. When the supply of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is limited by chlorate treatment, the synthesis machinery maintains sulfation of HS chains on basolateral SG-GFP until it is inhibited at 50 mM chlorate, whereas basolateral CS chains lose sulfate already at 12.5 mM chlorate and become longer. Apically, incorporation of 35S-sulfate into CS is reduced to a lesser extent at higher chlorate concentrations than basolateral CS, although apical CS is less intensely sulfated than basolateral CS in control cells. Similar to what was found for basolateral HS, sulfation of apical HS was not reduced at chlorate concentrations below 50 mM. Also, protein-free, xyloside-based GAG chains secreted basolaterally are more intensely sulfated than their apical counterpart, supporting the view that separate apical and basolateral pathways exist for GAG synthesis and sulfation. Introduction of benzyl beta-d-xyloside (BX) to the GAG synthesis machinery reduces the apical secretion of SG-GFP dramatically and also the modification of SG-GFP by HS.     

Protein core-dependent glycosaminoglycan modification and glycosaminoglycan-dependent polarized sorting in epithelial Madin-Darby canine kidney cells

The proteoglycan serglycin (SG) fused to green fluorescent protein (GFP) is secreted predominantly from the apical surface of polarized epithelial Madin-Darby canine kidney (MDCK) cell monolayers, but the minor fraction secreted basolaterally carries more intensely sulfated glycosaminoglycan (GAG) chains (Tveit H, Dick G, Skibeli V, Prydz K. 2005. A proteoglycan undergoes different modifications en route to the apical and basolateral surfaces of Madin-Darby canine kidney cells. J Biol Chem 280: 29596-29603). To investigate whether the domain with GAG attachment sites in SG (i) is sufficient to drive apical protein sorting and (ii) independently generates the sulfation differences observed in the apical and basolateral pathways, the GAG domain of SG was fused into the junction of rat growth hormone (rGH) and GFP and expressed in MDCK cells, either with or without two N-glycosylation sites in the rGH part. Both variants acquired chondroitin sulfate GAG chains and were secreted predominantly to the apical medium, to the same extent as rGH-GFP with two N-glycosylation sites only, and different from the nonsorted variant lacking glycosylation sites. Transfer of the GAG attachment domain from SG to the new rGH context abolished the differences in sulfation intensity and positions observed for SG in the apical and basolateral secretory routes. Thus, these differences are coded by elements outside the GAG attachment domain.     

N-Glycan synthesis in the apical and basolateral secretory pathway of epithelial MDCK cells and the influence of a glycosaminoglycan domain

Different classes of glycans are implicated as mediators of apical protein sorting in the secretory pathway of epithelial cells, but recent research indicates that sorting to the apical and basolateral surfaces may occur before completion of glycan synthesis. We have previously shown that a proteoglycan (PG) core protein can obtain different glycosaminoglycan (GAG) structures in the apical and basolateral secretory routes (Tveit H, Dick G, Skibeli V, Prydz K. 2005. A proteoglycan undergoes different modifications en route to the apical and basolateral surfaces of Madin-Darby canine kidney cells. J Biol Chem. 280:29596-29603) of epithelial Madin-Darby canine kidney (MDCK) cells. We have now also determined the detailed N-glycan structures acquired by a single glycoprotein species in the same apical and basolateral secretory pathways. For this purpose, rat growth hormone (rGH) with two N-glycan sites (rGH-2N) inserted into the rGH portion (NAS and NFT) was fused to green fluorescent protein (GFP) and expressed in MDCK cells. Immunoisolated rGH variants were analyzed for site occupancy and N-glycan structure by mass spectrometry. The extent of NAS and NFT site occupancy was different, but comparable for rGH-2N secreted apically and basolaterally. Microheterogeneity existed for the glycans attached to each N-glycan site, but no major differences were observed in the apical and basolateral pathways. Transfer of the GAG modification domain from the PG serglycin to the fusion site of rGH-2N and GFP allowed polymerization of GAG chains onto the novel protein variant and influenced the microheterogeneity of the N-glycans toward more acidic glycans, but did not alter the relative site occupancy. In conclusion, no major differences were observed for N-glycan structures obtained by the expressed model proteins in the apical and basolateral secretory pathways of epithelial MDCK cells, but insertion of a GAG attachment domain shifted the N-glycans to more acidic structures.     

Serglycin is a major proteoglycan in polarized human endothelial cells and is implicated in the secretion of the chemokine GROalpha/CXCL1

Proteoglycan (PG) expression was studied in primary human umbilical vein endothelial cells (HUVEC). RT-PCR analyses showed that the expression of the PG serglycin core protein was much higher than that of the extracellular matrix PG decorin and the cell surface PG syndecan-1. PG biosynthesis was further studied by biosynthetic [(35)S]sulfate labeling of polarized HUVEC. Interestingly, a major part of (35)S-PGs was secreted to the apical medium. A large portion of these PGs was trypsin-resistant, a typical feature of serglycin. The trypsin-resistant PGs were mainly of the chondroitin/dermatan sulfate type but also contained a minor heparan sulfate component. Secreted serglycin was identified by immunoprecipitation as a PG with a core protein of ～30 kDa. Serglycin was furthermore shown to be present in perinuclear regions and in two distinct types of vesicles throughout the cytoplasm using immunocytochemistry. To search for possible serglycin partner molecules, HUVEC were stained for the chemokine growth-related oncogene α (GROα/CXCL1). Co-localization with serglycin could be demonstrated, although not in all vesicles. Serglycin did not show overt co-localization with tissue-type plasminogen activator-positive vesicles. When PG biosynthesis was abrogated using benzyl-β-D-xyloside, serglycin secretion was decreased, and the number of vesicles with co-localized serglycin and GROα was reduced. The level of GROα in the apical medium was also reduced after xyloside treatment. Together, these findings indicate that serglycin is a major PG in human endothelial cells, mainly secreted to the apical medium and implicated in chemokine secretion.     

Dramatic changes of sulfated proteoglycans composition in a tumorigenic SV-40-transformed renal proximal-tubule cell line.

To characterize the sulfated proteoglycans (PGs) alterations associated with malignant transformation of epithelial cells in vitro, the localization, charge, size, and composition of cell-associated and secreted sulfated PGs have been compared in rabbit renal proximal-tubule cells in primary culture (Ronco et al., 1990) and in a derived SV-40 transformed cell line (RC.SV1) exhibiting a proximal phenotype and high tumor-inducing ability (Vandewalle et al., 1989). Both normal and transformed cells incorporated PGs into a thick basement membrane layer as shown by ruthenium red staining and immunodetection with a monoclonal antibody raised against the core protein of the bovine basement membrane heparan sulfate-PG (HS-PG). In primary cultures of normal cells, cell-associated PGs were almost identical to those extracted from renal tubule fractions in vivo by their size (Kav = 0.27 vs. 0.26 on Sepharose CL-6B) and composition characterized by the exclusive presence of heparan sulfate glycosaminoglycan (HS-GAG) chains. In addition, the cells secreted a HS-PG with similar biochemical characteristics (Kav = 0.29; 100% HS-GAG chains). The SV-40-transformed RC.SV1 cells also synthesized and secreted a unique PG with the same charge and Kav values and apparently the same core protein (35 kDa) as in nontransformed cells, but three major differences were observed: (i) an increased proportion of PG-associated [35S]sulfate radioactivity released into the culture medium (36 vs. 21%), (ii) the emergence of free GAG chains unincorporated into PGs and detected only in the cell-associated fraction, and (iii) a dramatic change in the composition of GAG chains in which chondroitin sulfate replaced heparan-sulfate. The latter finding is in keeping with the known chondroitin sulfate increase and heparan-sulfate decrease in epithelial tumors. The alterations of PGs observed in this study may play a role in the acquisition and/or maintenance of the malignant phenotype.     

Influenza M2 proton channel activity selectively inhibits trans-Golgi network release of apical membrane and secreted proteins in polarized Madin-Darby canine kidney cells

The function of acidification in protein sorting along the biosynthetic pathway has been difficult to elucidate, in part because reagents used to alter organelle pH affect all acidified compartments and are poorly reversible. We have used a novel approach to examine the role of acidification in protein sorting in polarized Madin-Darby canine kidney (MDCK) cells. We expressed the influenza virus M2 protein, an acid-activated ion channel that equilibrates lumenal and cytosolic pH, in polarized MDCK cells and examined the consequences on the targeting and delivery of apical and basolateral proteins. M2 activity affects the pH of only a subset of acidified organelles, and its activity can be rapidly reversed using ion channel blockers (Henkel, J.R., G. Apodaca, Y. Altschuler, S. Hardy, and O.A. Weisz. 1998. Mol. Biol. Cell. 8:2477-2490; Henkel, J.R., J.L. Popovich, G.A. Gibson, S.C. Watkins, and O.A. Weisz. 1999. J. Biol. Chem. 274:9854-9860). M2 expression significantly decreased the kinetics of cell surface delivery of the apical membrane protein influenza hemagglutinin, but not of the basolaterally delivered polymeric immunoglobulin receptor. Similarly, the kinetics of apical secretion of a soluble form of gamma-glutamyltranspeptidase were reduced with no effect on the basolaterally secreted fraction. Interestingly, M2 activity had no effect on the rate of secretion of a nonglycosylated protein (human growth hormone [hGH]) that was secreted equally from both surfaces. However, M2 slowed apical secretion of a glycosylated mutant of hGH that was secreted predominantly apically. Our results suggest a role for acidic trans-Golgi network pH in signal-mediated loading of apical cargo into forming vesicles.     

Cell density-dependent regulation of proteoglycan synthesis by transforming growth factor-beta(1) in cultured bovine aortic endothelial cells

The regulation of vascular endothelial cell behavior during angiogenesis and in disease by transforming growth factor-beta(1) (TGF-beta(1)) is complex, but it clearly involves growth factor-induced changes in extracellular matrix synthesis. Proteoglycans (PGs) synthesized by endothelial cells contribute to the formation of the vascular extracellular matrix and also influence cellular proliferation and migration. Since the effects of TGF-beta(1) on vascular smooth muscle cell growth are dependent on cell density, it is possible that TGF-beta(1) also directs different patterns of PG synthesis in endothelial cells at different cell densities. In the present study, dense and sparse cultures of bovine aortic endothelial cells were metabolically labeled with [(3)H]glucosamine, [(35)S]sulfate, or (35)S-labeled amino acids in the presence of TGF-beta(1). The labeled PGs were characterized by DEAE-Sephacel ion exchange chromatography and Sepharose CL-4B molecular sieve chromatography. The glycosaminoglycan M(r) and composition were analyzed by Sepharose CL-6B chromatography, and the core protein M(r) was analyzed by SDS-polyacrylamide gel electrophoresis, before and after digestion with papain, heparitinase, or chondroitin ABC lyase. These experiments indicate that the effect of TGF-beta(1) on vascular endothelial cell PG synthesis is dependent on cell density. Specifically, TGF-beta(1) induced an accumulation of small chondroitin/dermatan sulfate PGs (CS/DSPGs) with core proteins of approximately 50 kDa in the medium of both dense and sparse cultures, but a cell layer-associated heparan sulfate PG with a core protein size of approximately 400 kDa accumulated only in dense cultures. Moreover, only in the dense cell cultures did TGF-beta(1) cause CS/DSPG hydrodynamic size to increase, which was due to the synthesis of CS/DSPGs with longer glycosaminoglycan chains. The heparan sulfate PG and CS/DSPG core proteins were identified as perlecan and biglycan, respectively, by Western blot analysis. The present data suggest that TGF-beta(1) promotes the synthesis of both perlecan and biglycan when endothelial cell density is high, whereas only biglycan synthesis is stimulated when the cell density is low. Furthermore, glycosaminoglycan chains are elongated only in biglycan synthesized by the cells at a high cell density.     

Synthesis and release of sulfated glycoproteins by cultured glial cells

Both primary cultured glial cells and cloned (C-6) glioma cells have been shown to synthesize and release sulfated glycoproteins. It was found that N-linked tri- and tetra-antennary glycopeptides recovered from the glycoproteins contained most of the (35S) sulfate label. C-6 glial cells showed a higher rate of oligosaccharide sulfation than the primary glial cultures. Both cell types exhibited a high rate of release of sulfated glycoproteins into the medium. The ratio of 35S/3H incorporated from (35S) sulfate and (3H) glucosamine in the released material was higher than that of the glycoproteins associated with the cell, indicating an enrichment of sulfated glycoproteins in the secreted materials. Monensin inhibited both the synthesis and the release of sulfated glycoproteins.     

Association of Rab25 and Rab11a with the Apical Recycling System of Polarized Madin–Darby Canine Kidney Cells

Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin–Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways.     

Membrane-associated proteoglycans produced by Sertoli cells are not randomly distributed on the cell surface.

Sertoli cells in culture synthesize two membrane-associated proteoglycans (PGs) containing as glycosaminoglycan (GAG) moieties either chondroitin sulfate (CS) or CS and heparan sulfate (HS); the latter PG is, therefore, referred to as the mixed PG. To determine if these PGs are randomly distributed on the cell surface, Sertoli cell monolayers were treated with chondroitinase ABC, and then the remaining PGs were analyzed by DEAE-Sephacel chromatography. The results obtained with Sertoli cell monolayers show that the CS of the mixed PG is degraded by chondroitinase, while the CS-PG is not degraded. In contrast, chondroitinase treatment of Sertoli cells in suspension shows that both the mixed PG and the CS-PG are degraded. From this, it is inferred that the mixed PG is apically oriented and the CS-PG is basolaterally oriented. Studies of the adhesion of germ cells to Sertoli cell monolayers give further support to the apical location of the mixed PG and suggest that its HS moiety is involved in the attachment of germ cells to Sertoli cells.     

The role of an intracellular cysteine stretch in the sorting of the type II Na/phosphate cotransporter

The type II Na/phosphate cotransporters (NaPi-II) are critical for the control of plasma phosphate levels in vertebrates. NaPi-IIb mediates phosphate uptake from the small intestine followed by glomerular filtration and selective reabsorption from the renal proximal tubule by NaPi-IIa and NaPi-IIc. A C-terminal stretch of cysteine residues represents the hallmark of the NaPi-IIb isoforms. This motif is well conserved among NaPi-IIb type transporters but not found in other membrane proteins. To investigate the role of this motif we analyzed NaPi-II constructs in transiently and stably transfected MDCK cells. This cell line targets the NaPi-IIb isoforms from flounder and mouse to the apical membrane whereas the mouse IIa isoform shows no plasma membrane preference. Different parts of mouse NaPi-IIa and NaPi-IIb C-termini were fused to GFP-tagged flounder NaPi-II. The constructs showed strong staining of the plasma membrane with NaPi-IIb related constructs sorted predominantly apically, the IIa constructs localized apically and basolaterally with slight intracellular retention. When the cysteine stretch was inserted into the NaPi-IIa C-terminus, the construct was retained in a cytoplasmic compartment. 2-bromopalmitate, a specific palmitoylation inhibitor, released the transporter to apical and basolateral membranes. The drug also leads to a redistribution of the NaPi-IIb construct to both plasma membrane compartments. Immunoprecipitation of tagged NaPi-II constructs from [(3)H]-palmitate labeled MDCK cells indicated that the cysteine stretch is palmitoylated. Our results suggest that the modified cysteine motif prevents the constructs from basolateral sorting. Additional sorting determinants located downstream of the cysteine stretch may release the cargo to the apical compartment.     

Synthesis of sulfated glycoproteins by glial precursor cells

Populations of enriched glial precursor cells and astrocytes isolated from primary cultures of newborn rat brain were used to study the synthesis of sulfated glycoproteins. Both cell types incorporated [³H]glucosamine and [³⁵S]sulfate into carbohydrate side chains of proteoglycans and glycoproteins. The rate of incorporation of [³H]glucosamine into the oligosaccharides and the pattern of distribution of the label into high mannose and complex glycopeptides recovered from the glycoproteins appeared to be similar for the two glial cell types. However, clear differences were noted in the rate of oligosaccharide sulfation activities. Thus the cultures of precursor glia were about four times more active than cultures enriched in astroglia in their ability to incorporate [³⁵S]sulfate into glycoproteins.     

Influence of intermittent compressive force on proteoglycan content in calcifying growth plate cartilage in vitro

We investigated the effect of mechanical stimulation by an intermittent compressive force (ICF) on proteoglycan (PG) synthesis and PG structure in calcified and noncalcified cartilage of fetal mouse long bone rudiments. Uncalcified cartilaginous long bone rudiments were cultured for 5 days in the presence of [35S]sulfate and [3H]glucosamine under control conditions (atmospheric pressure) or under the influence of ICF. ICF was generated by intermittently compressing the gas phase above the culture medium (130 mbar, 0.3 Hz). During culture, the center of the rudiments started to calcify. ICF stimulated calcification such that, after 5 days, the diaphysis of calcified cartilage was about two times as long as in the control cultures. At the end of the experiment, the rudiments were divided in a central calcified diaphysis and two noncalcified epiphyses. Diaphysis and epiphyses were pooled separately. PGs were extracted with 4 M guanidinium chloride and isolated by cesium chloride density gradient centrifugation. PGs (predigested with proteinase K or chondroitinase ABC) were characterized for hydrodynamic size of aggregates, monomers, and chondroitin sulfate chains by gel permeation chromatography and for degree of sulfation by ion exchange chromatography on high pressure liquid chromatography columns. ICF increased the amount of incorporated sulfate per tissue volume unit in the noncalcified epiphyses, but decreased this parameter in the calcified diaphysis. However, in both calcified and noncalcified cartilage, ICF increased the degree of sulfation of the chondroitin sulfate chains. No effects were found on the hydrodynamic size of the PG aggregates or monomers, but in the epiphyses ICF increased the size of the chondroitin sulfate chains. No other changes of structural characteristics of the macromolecules were observed. This study demonstrates that ICF generally stimulated the incorporation of [35S]sulfate into chondroitin sulfate chains. We conclude from the lowered [35S]sulfate content in calcified cartilage that ICF reduced the number of chondroitin sulfate chains and probably PGs while accelerating matrix calcification. It seems likely that the two effects are linked, indicating that a reduction of the number of chondroitin sulfate chains is part of the complicated process of cartilage calcification.     

N-glycans are not a universal signal for apical sorting of secretory proteins.

In MDCK cells, N-glycans have been shown to determine the sorting of secretory proteins and membrane proteins to the apical domain in the absence of a dominant basolateral targeting signal. We have examined the sorting of endogenous proteins in ECV304 cells in the presence and absence of tunicamycin, an inhibitor of N-linked glycosylation. A prominent apically secreted protein of 71 kDa was not N-glycosylated and continued to be secreted apically in the presence of tunicamycin. In contrast, other endogenous proteins that were N-glycosylated were secreted preferentially into the basolateral medium or without polarity. When rat growth hormone was expressed in MDCK and ECV304 cells, we observed 65 and 94% of the secretion to the basolateral medium, respectively. Introduction of a single N-glycan caused 83% of the growth hormone to be secreted at the apical surface in MDCK cells but had no significant effect on the polarity of secretion of growth hormone in ECV304 cells. These results indicate that not all cell lines recognise N-glycans as a signal for apical sorting and raises the possibility of using ECV304 cells as a model system for analysis of apical sorting molecules.     

Apical secretion and sialylation of soluble dipeptidyl peptidase IV are two related events

The role of glycans in the apical targeting of proteins in epithelial cells remains a debated question. We have expressed the mouse soluble dipeptidyl peptidase IV (DPP IV ectodomain) in kidney (MDCK) and in intestinal (Caco-2) epithelial cell lines, as a model to study the role of glycosylation in apical targeting. The mouse DPP IV ectodomain was secreted mainly into the apical medium by MDCK cells. Exposure of MDCK cells to GalNac-alpha-O-benzyl, a drug previously described as an inhibitor of mucin O-glycosylation, produced a protein with a lower molecular weight. In addition this treatment resulted in a decreased apical secretion and an increased basolateral secretion of mouse DPP IV ectodomain. When expressed in Caco-2 cells, the mouse DPP IV ectodomain was secreted mainly into the basolateral medium. However, BGN was still able to decrease the amount of apically secreted protein and to increase its basolateral secretion. Neuraminidase digestion showed that the most striking effect of BGN was a blockade of DPP IV sialylation in both MDCK and Caco-2 cells. These results indicate that a specific glycosylation step, namely, sialylation, plays a key role in the control of the apical targeting of a secreted DPP IV both in MDCK and Caco-2 cells.     

Glycosylphosphatidylinositol-anchored proteins are preferentially targeted to the basolateral surface in Fischer rat thyroid epithelial cells

Glycosylphosphatidylinositol (GPI) acts as an apical targeting signal in MDCK cells and other kidney and intestinal cell lines. In striking contrast with these model polarized cell lines, we show here that Fischer rat thyroid (FRT) epithelial cells do not display a preferential apical distribution of GPI-anchored proteins. Six out of nine detectable endogenous GPI-anchored proteins were localized on the basolateral surface, whereas two others were apical and one was not polarized. Transfection of several model GPI proteins, previously shown to be apically targeted in MDCK cells, also led to unexpected results. While the ectodomain of decay accelerating factor (DAF) was apically secreted, 50% of the native, GPI-anchored form, of this protein was basolateral. Addition of a GPI anchor to the ectodomain of Herpes simplex gD-1, secreted without polarity, led to basolateral localization of the fusion protein, gD1-DAF. Targeting experiments demonstrated that gD1-DAF was delivered vectorially from the Golgi apparatus to the basolateral surface. These results indicate that FRT cells have fundamental differences with MDCK cells with regard to the mechanisms for sorting GPI-anchored proteins: GPI is not an apical signal but, rather, it behaves as a basolateral signal. The "mutant" behavior of FRT cells may provide clues to the nature of the mechanisms that sort GPI-anchored proteins in epithelial cells.     

Hepatocyte growth factor inhibits intrinsic antibacterial activity of Madin-Darby canine kidney cells

We investigated whether or not polarized renal epithelial cells produce antibacterial factors, which aid in host defense at the cell surface of renal epithelium. A model of polarized Madin-Darby canine kidney (MDCK) epithelial cells grown on filters was used to test for the presence of apically or basolaterally secreted factors on the growth of non-virulent (XL1-Blue) and uropathogenic (J96) strains of Escherichia coli (E. coli). Growth of both XL1-Blue and J96 strains of E. coli in medium on the apical and basolateral surface of MDCK cells was inhibited as compared to bacterial growth in medium not exposed to MDCK cells. The inhibition of bacterial growth was similar in both apical and basolateral surface medium. Pretreatment of MDCK cells with hepatocyte growth factor (HGF) blunted the inhibition of XL1-Blue and J96 growth in apical and basolateral surface medium as compared to growth in medium on the surfaces of untreated MDCK cells. Immunofluorescent analysis demonstrated the presence of beta-defensin isoforms 1-3 in MDCK cells, with isoform 1 being the most prevalent form observed. HGF treatment reduced the amount of immunoreactive beta-defensin-1 in MDCK cells. These data demonstrate that polarized renal epithelium produce antibacterial factors. The renotropic growth factor HGF inhibits these antibacterial factors. beta-defensins may contribute to this antibacterial activity and play an important role in renal epithelial resistance to bacterial infections.     

Characterization of proteoglycans synthesized by rat thyroid cells in culture and their response to thyroid-stimulating hormone

A Fisher rat thyroid cell line was maintained in culture and the cells were labeled with [3H]glucosamine, [35S]sulfate, and [35S]cysteine to examine the synthesis of proteoglycans. 3H and 35S radioactivity from these precursors were incorporated into both chondroitin sulfate (CS) and heparan sulfate (HS) proteoglycans. CS proteoglycans were almost exclusively secreted into the medium while HS proteoglycans remained mainly associated with the cell layer. Single chain glycosaminoglycans released by papain digestion or alkaline borohydride treatment of either the CS or HS proteoglycans had average molecular weights of approximately 30,000 on Sepharose CL-6B chromatography. Both CS and HS proteoglycans were relatively small and contained only one or two glycosaminoglycans chains. 3H and 35S incorporation into both CS and HS proteoglycans were increased by thyroid-stimulating hormone (TSH) in a dose-dependent manner, which is in part explained by an adenylate cyclase-dependent mechanism as indicated by a similar effect in response to dibutyryl cAMP. TSH enhanced the incorporation of 35S into CS from [35S]cysteine about 1.5-fold and that from [35S]sulfate about 2-fold. This result demonstrated that the increased 35S incorporation from the [35S]sulfate precursor reflects an actual increase in sulfate incorporation and is not simply a result from an apparent increase in specific activity of the phosphoadenosine phosphosulfate donor. Analysis of disaccharides from chondroitinase digests revealed that the proportion of non-sulfated, 4-sulfated, and 6-sulfated disaccharides was not altered appreciably by TSH. These results, together with the disproportionate increase in 3H incorporation into CS from [3H]glucosamine, indicated that TSH increased the specific activity of the 3H label as well. Chase experiments revealed that CS proteoglycans were rapidly (t1/2 = 15 min) secreted into the medium and that the degradation of cell-associated proteoglycans was enhanced by TSH.     

High-glucose-induced structural changes in the heparan sulfate proteoglycan, perlecan, of cultured human aortic endothelial cells

Hyperglycemia is an independent risk factor for diabetes-associated cardiovascular disease. One potential mechanism involves hyperglycemia-induced changes in arterial wall extracellular matrix components leading to increased atherosclerosis susceptibility. A decrease in heparan sulfate (HS) glycosaminoglycans (GAG) has been reported in diabetic arteries. The present studies examined the effects of high glucose on in vitro production of proteoglycans (PG) by aortic endothelial cells. Exposure of cells to high glucose (30 vs. 5 mM glucose) resulted in decreased [(35)S] sodium sulfate incorporation specifically into secreted HSPG. Differences were not due to hyperosmolar effects and no changes were observed in CS/DSPG. Enzymatic procedures, immunoprecipitation and Western analyses demonstrated that high glucose induced changes specifically in the HSPG, perlecan. In double-label experiments, lower sulfate incorporation in high-glucose-treated cells was accompanied by lower [(3)H] glucosamine incorporation into GAG but not lower [(3)H] serine incorporation into PG core proteins. Size exclusion chromatography demonstrated that GAG size was unchanged and GAG sulfation was not reduced. These results indicate that the level of regulation of perlecan by high glucose is posttranslational, involving a modification in molecular structure, possibly a decrease in the number of HS GAG chains on the core protein.     

Secretory cargo composition affects polarized secretion in MDCK epithelial cells

Polarized epithelial cells secrete proteins at either the apical or basolateral cell surface. A number of non-epithelial secretory proteins also exhibit polarized secretion when they are expressed in polarized epithelial cells but it is difficult to predict where foreign proteins will be secreted in epithelial cells. The question is of interest since secretory epithelia are considered as target tissues for gene therapy protocols that aim to express therapeutic secretory proteins. In the parathyroid gland, parathyroid hormone is processed by furin and co-stored with chromogranin A in secretory granules. To test the secretion of these proteins in epithelial cells, they were expressed in MDCK cells. Chromogranin A and a secreted form of furin were secreted apically while parathyroid hormone was secreted 60% basolaterally. However, in the presence of chromogranin A, the secretion of parathyroid hormone was 65% apical, suggesting that chromogranin can act as a “sorting escort” (sorting chaperone) for parathyroid hormone. Conversely, apically secreted furin did not affect the sorting of parathyroid hormone. The apical secretion of chromogranin A was dependent on cholesterol, suggesting that this protein uses an established cellular sorting mechanism for apical secretion. However, this sorting does not involve the N-terminal membrane-binding domain of chromogranin A. These results suggest that foreign secretory proteins can be used as “sorting escorts” to direct secretory proteins to the apical secretory pathway without altering the primary structure of the secreted protein. Such a system may be of use in the targeted expression of secretory proteins from epithelial cells. David V. Cohn—Deceased.