Antisense RNA Strategy Research Articles

Upregulation of MMP-2 by all-trans retinoic acid is mediated by TGF- β 1 in cultured rat mesangial cell

Matrix metalloproteinase-2 (MMP-2) degrades basement membrane collagen and its abnormal expression is associated with glomerulonephritis and glomerulosclerosis. All-trans retinoic acid (ATRA) has been indicated as preventing age-related glomerulosclerosis. The results of our study showed that ATRA upregulated expression of MMP-2 mRNA and secretion of MMP-2 proteins, and increased enzymatic activity of MMP-2 in cultured mesangial cell. In addition, ATRA also caused a dose-dependent increase in the secretion of transforming growth factor-β (TGF-β1) peptides. Since TGF-β1 regulated the expression of MMP-2 in mesangial cell, it is possible that TGF-β1 is involved in ATRA-induced MMP-2 expression. Using antisense TGF-β1 RNA strategy, antisense TGF-β1 construct almost completely blocked secretion of active TGF-β1 peptides in the antisense-bearing transfectants. Northern blot analysis showed that the transfectants treated with 10–6M ATRA for 72 h neither increased nor decreased the levels of MMP-2 messenger ribonucleic acid (mRNA). Moreover, addition of anti-TGF-β1 antibodies to mesangial cell in the presence of 10–6M ATRA reduced ATRA-induced MMP-2 expression dose dependently. These data suggest that TGF-β1 might be involved in ATRA-induced MMP-2 expression.

An explosive antisense RNA strategy for inhibition of a lactococcal bacteriophage.

The coding regions of six putative open reading frames (ORFs) identified near the phage phi31 late promoter and the right cohesive end (cos) of lactococcal bacteriophage phi31 were used to develop antisense constructs to inhibit the proliferation of phage phi31. Two middle-expressed ORFs (ORF 1 and ORF 2) and four late-expressed ORFs (ORF 3 through ORF 6) were cloned individually between the strong Lactobacillus P6 promoter and the T7 terminator (T(T7)) to yield a series of antisense RNA transcripts. When expressed on a high-copy-number vector from a strong promoter, the constructs had no effect on the efficiency of plaquing (EOP) or the plaque size of phage phi31. To increase the ratio of antisense RNA to the targeted sense mRNA appearing during a phage infection, the antisense cassettes containing the late-expressed ORFs (ORF 3 through ORF 6) were subcloned to pTRK360, a low-copy-number vector containing the phage phi31 origin of replication, ori31. ori31 allows for explosive amplification of the low-copy-number vector upon phage infection, thereby increasing levels of antisense RNA transcripts later in the lytic cycle. In addition, the presence of ori31 also lowers the burst size of phage phi31 fourfold, resulting in fewer sense, target mRNAs being expressed from the phage genome. The combination of ori31 and P6::anti-ORF 4H::T(T7) resulted in a threefold decrease in the EOP of phage phi31 (EOP = 0.11 +/- 0.03 [mean +/- standard deviation]) compared to the presence of ori31 alone (EOP = 0.36). One-step growth curves showed that expression of anti-ORF 4H RNA decreased the percentage of successful centers of infection (75 to 80% for ori31 compared to 35 to 45% for ori31 plus anti-ORF 4H), with no further reduction in burst size. Growth curves performed in the presence of varying levels of phage phi31 showed that ori31 plus anti-ORF 4H offered significant protection to Lactococcus lactis, even at multiplicities of infection of 0.01 and 0.1. These results illustrate a successful application of an antisense strategy to inhibit phage replication in the wake of recent unsuccessful reports.

Adenovirus-Based Phospholamban Antisense Expression as a Novel Approach to Improve Cardiac Contractile Dysfunction

A decrease in sarcoplasmic reticulum Ca(2+) pump (SERCA2) activity is believed to play a role in the impairment of diastolic function of the failing heart. Because the expression ratio of phospholamban (PL) to SERCA2 may be a target to improve contractile dysfunction, a PL antisense RNA strategy was developed under the control of either a constitutive cytomegalovirus (CMV) or an inducible atrial natriuretic factor (ANF) promoter. The latter is upregulated in hypertrophied and failing heart, allowing "induction-by-disease" gene therapy. Part of the PL cDNA was cloned in antisense and sense directions into adenovectors under the control of either a CMV (Ad5CMVPLas and Ad5CMVPLs, respectively) or ANF (Ad5ANFPLas and Ad5ANFPLs, respectively) promoter. Infection of cultured rat neonatal cardiomyocytes with Ad5CMVPLas reduced PL mRNA to 30+/-7% of baseline and PL protein to 24+/-3% within 48 and 72 hours, respectively. The effects were vector dose dependent. Ad5CMVPLas increased the Ca(2+) sensitivity of SERCA2 and reduced the time to 50% recovery of the Ca(2+) transient. A decrease of PL protein was also achieved by infection with Ad5ANFPLas, and the presence of the hypertrophic stimulus, endothelin-1, led to enhanced downregulation of PL. The adenovectors expressing PL sense RNA had no effect on any of the tested parameters. Vector-mediated PL antisense RNA expression may become a feasible approach to modulate myocyte Ca(2+) homeostasis in the failing heart. The inducible ANF promoter for the first time offers the perspective for induction-by-disease gene therapy, ie, selective expression of therapeutic genes in hypertrophied and failing cardiomyocytes.

Stress-induced membrane association of the Streptococcus mutans GTP-binding protein, an essential G protein, and investigation of its physiological role by utilizing an antisense RNA strategy.

SGP (for Streptococcus GTP-binding protein) is a Streptococcus mutans essential GTPase which has significant sequence identity to the previously identified Escherichia coli Era protein and to numerous other prokaryotic GTPase proteins of unknown function. Recent studies in our laboratory have addressed the possible role of SGP in the stress response of the oral pathogen S. mutans. Here we report that during growth in the early stationary phase, and in response to elevated temperatures or acidic pH, the distribution of SGP between the cytoplasm and the membranes of S. mutans cells varies. Immunoblot analysis of soluble and membrane protein fractions collected from the mid-log and early stationary growth phases of bacterial populations grown at normal temperature (37 degrees C) and at the elevated temperature of 43 degrees C, or at acidic pH, demonstrated that the total amount of SGP increased with the age of the bacterial culture, elevated temperature, or acidic pH. Furthermore, it was established that a substantial amount of SGP is associated with the membrane fraction under stress conditions. In order to investigate the physiological role of SGP, we constructed an S. mutans strain capable of chromosomal sgp antisense RNA expression, which interferes with the normal information processing of the sgp gene. Utilizing this strain, we determined conditions whereby the streptococcal cells can be depleted of SGP, thus avoiding the problem of constructing a conditional lethal system. From the results of measurements of the nucleotide pools extracted from the antisense strain and its isogenic counterpart, we propose that one of the physiological roles of SGP is regulation and modulation of the GTP/GDP ratio under different growth conditions. Moreover, we observed that in SGP-depleted cells the levels of glucan-binding protein A (GbpA) substantially increased, suggesting that GbpA may have stress response-related physiological functions. Finally, the potential applications of the antisense RNA approach that we employed are discussed.

Metabolic Flux Analysis Elucidates the Importance of the Acid-Formation Pathways in Regulating Solvent Production by Clostridium acetobutylicum

Metabolic flux analysis was used to investigate the roles of the acid formation pathways in Clostridium acetobutylicum. The acid formation pathways were revealed to serve different roles in wildtype fermentations than previously expected. Specifically, enzymes known to catalyze butyrate formation were found to uptake butyrate without concomitant production of acetone. This role was further corroborated by flux analysis of a recombinant strain overexpressing the butyrate formation enzymes. Analysis of wildtype fermentation data also revealed an important role for the acetate formation enzymes, namely the cycling of carbon between acetate and acetyl-CoA during the stationary phase. Next, metabolic flux analysis was used to compare the patterns of activity in two butyrate kinase deficient strains of C. acetobutylicum. The strain developed by gene inactivation, PJC4BK, exhibited a shift in acid formation fluxes toward acetate while the strain developed by antisense RNA strategies, 824(pRD4), did not exhibit such a shift. However, both strains exhibited altered solvent formation patterns. PJC4BK exhibited a strong transient enhancement of solvent formation fluxes. In contrast, 824(pRD4) exhibited relatively lower levels of solvent formation fluxes, although fluxes were sustained over a longer period of time.

CREB mediates the cAMP-responsiveness of the tyrosine hydroxylase gene: use of an antisense RNA strategy to produce CREB-deficient PC12 cell lines

cAMP initiates the PKA signaling cascade in rat pheochromocytoma PC12 cells, resulting in transcriptional activation of the tyrosine hydroxylase (TH) gene. This effect is mediated primarily through the cAMP responsive element (CRE), located at position −45 to −38 within the TH gene promoter. In this study, we applied an antisense RNA strategy to evaluate the role of the cAMP responsive element binding protein (CREB) in regulating TH gene expression. CREB antisense RNA expression vectors were stably introduced into PC12 cells to generate cell lines deficient in CREB. CREB protein and mRNA levels were diminished up to 90% in the stably transfected cell lines. Promoter analysis experiments demonstrated that cAMP-mediated inducibility of either TH gene proximal promoter activity or the activity of the TH CRE by itself fused upstream of a basal promoter was diminished in CREB-deficient cell lines. PKA activity in the CREB-deficient cell lines was comparable to the activity in control cell lines. In addition, neither ATF1, nor CREM proteins were significantly down-regulated in the CREB-deficient cells. Most significantly, the cAMP-inducibility of endogenous TH mRNA was completely blocked in the CREB-deficient cells, indicating that the response of the endogenous gene to cAMP was dependent on CREB. These results support the hypothesis that CREB (not other CRE-binding proteins) is the key transcription factor that is required for regulating TH gene expression in response to cAMP. Furthermore, our studies indicate that these CREB-deficient PC12 cells are excellent tools to study the participation of CREB in gene regulation.

Reversible tumorigenesis induced by deficiency of vasodilator-stimulated phosphoprotein.

Random homozygous knockout (RHKO) is an antisense RNA strategy capable of identifying genes whose homozygous functional inactivation yields a selectable phenotype in cells growing in culture. Using this approach, we isolated NIH 3T3 fibroblast clones that showed the ability to form colonies on 0.5% agar and tumors in nude mice. The gene inactivated in one of these clones was found to encode VASP (vasodilator-stimulated phosphoprotein), a previously identified protein that binds to components of the cadherin-catenin junctional complex and has been implicated in cell-cell interactions, the formation of actin filaments, and the transmission of signals at the cytoskeleton-membrane interface. Fibroblasts made deficient in VASP by RHKO showed loss of contact inhibition, and consequently, continued cell division past confluence. Restoration of VASP function by reversal of RHKO yielded cells that had lost the neoplastic capabilities acquired during RHKO. Overproduction of VASP mRNA in the sense or antisense orientation from expression constructs introduced by transfection into naive NIH 3T3 fibroblasts also resulted in neoplastic transformation, implying that normal cell growth may require the maintenance of VASP expression within a narrow range. Our results implicate VASP in tumorigenesis and/or cancer progression.

Antisense RNA strategies for metabolic engineering of Clostridium acetobutylicum.

We examined the effectiveness of antisense RNA (as RNA) strategies for metabolic engineering of Clostridium acetobutylicum. Strain ATCC 824(pRD4) was developed to produce a 102-nucleotide asRNA with 87% complementarity to the butyrate kinase (BK) gene. Strain ATCC 824(pRD4) exhibited 85 to 90% lower BK and acetate kinase specific activities than the control strain. Strain ATCC 824(pRD4) also exhibited 45 to 50% lower phosphotransbutyrylase (PTB) and phosphotransacetylase specific activities than the control strain. This strain exhibited earlier induction of solventogenesis, which resulted in 50 and 35% higher final concentrations of acetone and butanol, respectively, than the concentrations in the control. Strain ATCC 824(pRD1) was developed to putatively produce a 698-nucleotide asRNA with 96% complementarity to the PTB gene. Strain ATCC 824(pRD1) exhibited 70 and 80% lower PTB and BK activities, respectively, than the control exhibited. It also exhibited 300% higher levels of a lactate dehydrogenase activity than the control exhibited. The growth yields of ATCC 824(pRD1) were 28% less than the growth yields of the control. While the levels of acids were not affected in ATCC 824(pRD1) fermentations, the acetone and butanol concentrations were 96 and 75% lower, respectively, than the concentrations in the control fermentations. The lower level of solvent production by ATCC 824(pRD1) was compensated for by approximately 100-fold higher levels of lactate production. The lack of any significant impact on butyrate formation fluxes by the lower PTB and BK levels suggests that butyrate formation fluxes are not controlled by the levels of the butyrate formation enzymes.

Reply from H-U. Koop, W. Kofer and K. Steinmüller

Reply from H-U. Koop, W. Kofer and K. Steinmüller

The sgp gene modulates stress responses of Streptococcus mutans: utilization of an antisense RNA strategy to investigate essential gene functions

The sgp gene from Streptococcus mutans has been previously isolated, characterized, and demonstrated to encode a G-protein. In order to investigate the function of this gene, a novel antisense RNA strategy was developed. Expression of sgp antisense RNA in Escherichia coli led to transient inhibition of growth. In addition, sgp antisense RNA expression in S. mutans resulted in decreased growth under environmental stress conditions (44°C, acidic pH, and high osmolarity). Therefore, these results suggest that the sgp gene plays a role in modulating the stress responses of S. mutans. This approach could be applicable for investigating the function of essential genes in other organisms for which mutants are not available.

The sgp gene modulates stress responses of Streptococcus mutans: utilization of an antisense RNA strategy to investigate essential gene functions.

The sgp gene from Streptococcus mutans has been previously isolated, characterized, and demonstrated to encode a G-protein. In order to investigate the function of this gene, a novel antisense RNA strategy was developed. Expression of sgp antisense RNA in Escherichia coli led to transient inhibition of growth. In addition, sgp antisense RNA expression in S. mutans resulted in decreased growth under environmental stress conditions (44 degrees C, acidic pH, and high osmolarity). Therefore, these results suggest that the sgp gene plays a role in modulating the stress responses of S. mutans. This approach could be applicable for investigating the function of essential genes in other organisms for which mutants are not available.

Ex vivo and in vivo IGF-I antisense RNA strategies for treatment of cancer in humans.

Two technical approaches to gene therapy for cancer utilize ex vivo and in vivo gene transfer methodology. This paper focuses on applicability and use of each of these approaches using an IGF-I antisense RNA strategy of treatment. Insulin-like growth factor I (IGF-I) and IGF-II have pivotal roles in cell proliferation and development (for review see 1–4). The preponderance of peptide synthesis and activity occur during fetal development. Protein synthesis is down-regulated in most mature tissues except for adult liver. However, many cancers produce and secrete IGF-I and/or IGF-II and also express IGF-I receptor. These include: astrocytomas, glioblastoma multiforme, sarcomas, thyroid adenomas, hepatocellular cancer, small cell cancer of the lung, and carcinomas of breast, colon, and prostate gland. The initiation and/or early growth of these tumors may be dependent upon changes in these mitogenic peptides or their receptors. Kaleko et. al. (1990) showed that overexpression of human IGF-I receptor in mice fibroblasts promotes colony formation in soft agar for cells grown in 10% serum supplemented with IGF-I. Trojan, Ilan, and colleagues demonstrated in 1992 that inhibition of IGF-I expression by an IGF-I antisense RNA strategy of gene therapy inhibits tumor formation and causes regression and cure of wild-type C6 glioblastoma in rats (6). They further showed that tumor was infiltrated with CD8 lymphocytes as the process resolved, suggesting that the treatment may act through an immunogenic mechanism (7). Resnicoff, Baserga, and colleagues showed that down-regulation of the IGF-I receptor in rat C6 glioblastoma cells by an antisense strategy also prevents tumor formation and/or causes regression of tumors induced by wild type C6 cells (8–9).

Decreased expression of the high-mobility group protein T160 by antisense RNA impairs the growth of mouse fibroblasts

The T160 protein belongs to the HMG-1 box protein family and preferentially binds to non-B-DNA conformations with no sequence specificity. Its exacts role has yet to be defined, though it seems to participate in processes involving DNA, such as replication, transcription and recombination. We have used an antisense RNA strategy to investigate its role in cell growth and proliferation. T160 expression is strongly suppressed by stable introduction of an antisense construct into N1H3T3 cells, and this decrease is accompanied by substantial changes in the growth properties of the stable transfectants. Impaired growth of T160 − cells was mainly related to two mechanisms: i) decreased rates of cell proliferation at normal serum concentration: and ii) occurrence of cell death by apoptosis at low serum concentration, as demonstrated by both flow cytometry and microscopy. The finding that decreased T160 availability affects cell proliferation, provides further evidence of its involvement in a basic cell function, such as DNA replication.

Antiviral potency of drug-gene therapy combinations against human immunodeficiency virus type 1.

Gene therapy for the treatment of human immunodeficiency virus type 1 (HIV-1) infection using intracellular immunization strategies is currently being tested in clinical trials. With the continuing development of potent antiretroviral drugs (e.g., reverse transcriptase [RT] and protease [PR] inhibitors), it is likely that HIV-1 gene therapy will be applied to humans concurrently receiving such antiretroviral medication. In this study, we assessed the in vitro antiviral efficacy of two gene therapy strategies (trans-dominant RevM10, Gag antisense RNA) in combination with clinically relevant RT (AZT, ddC) or PR (indinavir) inhibitors. Retrovirally transduced, human T cell lines expressing antiviral gene constructs were inoculated with high doses of HIV-1HXB3 in the presence or absence of inhibitors. The combination of RevM10 or Gag antisense RNA with antiviral drugs inhibited HIV-1 replication 10-fold more effectively than the single antiviral drug regimen alone. More importantly, we also addressed whether gene therapy strategies are effective against drug-resistant HIV-1 isolates. Both the RevM10 and Gag antisense RNA strategies showed antiviral efficacy against several RT inhibitor-resistant HIV-1 isolates equivalent to their inhibition of HIV-1HXB3 replication. In summary, our data demonstrate the greater than additive antiviral efficacy of gene therapy strategies and RT or PR inhibitors, and that gene therapy approaches are effective against drug-resistant HIV-1 viral isolates.

Expression of a D 2 dopamine receptor antisense RNA in brain inhibits D 2-mediated behaviors

Drugs currently used to treat disorders of dopamine-mediated behaviors in the central nervous system are non-selective in that they interact not only with more than one isoform of dopamine receptor but also with receptors for other neurotransmitters. A new strategy to inhibit the actions of individuals dopamine receptor subtypes is to inhibit the synthesis of the receptors through the use of oligonucleotides antisense to the transcripts encoding the different receptors. Earlier studies showed that oligodeoxynucleotides antisense to the D 1 or D 2 dopamine receptor messenger RNAs specifically inhibited the biological actions mediated by these individual isoforms of the dopamine receptor. However, these actions were relatively short-lasting. To determine whether one can achieve long-lasting inhibition of dopamine responses, while still taking advantage of the highly selective nature of an antisense strategy, an expression vector was employed that generates antisense RNA to the transcripts encoding the D 2 dopamine receptor. A single intrastriatal injection of this vector generated an antisense RNA to the D 2 dopamine receptor, selectively reduced the levels of D 2 dopamine receptors, and caused selective, long-term inhibition of behaviors mediated by D 2 dopamine agonists. Such an antisense RNA strategy may find use in studying the function of dopaminergic receptors and in disorders associated with dopaminergic hyperactivity.

Antisense basic fibroblast growth factor gene transfer reduces neointimal thickening after arterial injury.

To determine whether synthesis of endogenous basic fibroblast growth factor (bFGF) after arterial injury is critical to the intimal thickening response, intraluminal adenoviral gene transfer of an antisense bFGF (Ad.ASbFGF) transgene was used to inhibit the subsequent synthesis of bFGF protein after injury. Sprague-Dawley rats underwent balloon catheter carotid artery injury and in vivo gene transfer. Isolated segments of rat common carotid artery were infected with an adenoviral vector encoding an antisense bFGF transcript at concentrations of 2 x 10(9), 1 x 10(10), or 1 x 10(11) pfu/ml. Control rats were treated with either a control adenovirus encoding the beta-galactosidase gene, (Ad.lacZ), at 1 x 10(10), or 1 x 10(11) pfu/ml, or phosphate-buffered saline solution (vehicle). Two weeks after injury the rats were killed and perfusion-fixed. Cross-sectional areas of the carotid arterial intima and media were measured by planimetry, and the intima/media ratio (I/M) was calculated for each vessel. The mean I/M for each Ad.ASbFGF group and controls were compared and the significance assessed by analysis of variance. At two weeks after injury, the highest dose of Ad.ASbFGF, 1 x 10(11) pfu/ml, resulted in a near total inhibition of thickening (I/M = 0.14 +/- 0.04, mean +/- SEM) when compared with phosphate-buffered saline solution alone (I/M = 0.99 +/- 0.07), or Ad.lacZ 1 x 10(10) pfu/ml (I/M = 1.01 +/- 0.10) control treatments (p < 0.01). A tenfold lower dose of Ad.ASbFGF, 1 x 10(10) pfu/ml, also caused significant reduction in intimal thickening (I/M = 0.39 +/- 0.07, p < 0.01). Treatment with 2 x 10(9) pfu/ml Ad.ASbFGF did not significantly limit intimal thickening (I/M = 0.72 +/- 0.12). Inhibition of bFGF synthesis in vivo using an antisense RNA strategy significantly inhibits intimal thickening after arterial balloon injury. This study suggests that continued bFGF synthesis contributes to intimal thickening after arterial injury, and that antisense bFGF may represent an effective strategy in limiting restenosis after angioplasty.

Inhibition of laminin alpha 1-chain expression leads to alteration of basement membrane assembly and cell differentiation.

The expression of the constituent alpha 1 chain of laminin-1, a major component of basement membranes, is markedly regulated during development and differentiation. We have designed an antisense RNA strategy to analyze the direct involvement of the alpha 1 chain in laminin assembly, basement membrane formation, and cell differentiation. We report that the absence of alpha 1-chain expression, resulting from the stable transfection of the human colonic cancer Caco2 cells with an eukaryotic expression vector comprising a cDNA fragment of the alpha 1 chain inserted in an antisense orientation, led to (a) an incorrect secretion of the two other constituent chains of laminin-1, the beta 1/gamma 1 chains, (b) the lack of basement membrane assembly when Caco2-deficient cells were cultured on top of fibroblasts, assessed by the absence of collagen IV and nidogen deposition, and (c) changes in the structural polarity of cells accompanied by the inhibition of an apical digestive enzyme, sucrase-isomaltase. The results demonstrate that the alpha 1 chain is required for secretion of laminin-1 and for the assembly of basement membrane network. Furthermore, expression of the laminin alpha 1-chain gene may be a regulatory element in determining cell differentiation.

Rice allergenic protein and molecular-genetic approach for hypoallergenic rice.

Allergenic proteins with a molecular mass of about 14 to 16 kDa were isolated from a rice salt-soluble fraction based on the reactivity with IgE antibodies from patients allergic to rice. cDNA clones encoding these allergenic proteins were isolated from a cDNA library of maturing rice seeds, and the deduced amino acid sequences showed considerable similarity to wheat and barley alpha-amylase/trypsin inhibitors, which have recently been identified as major allergens associated with baker's asthma. An antisense RNA strategy was applied to repress the allergen gene expression in maturing rice seeds. Immunoblotting and ELISA analyses of the seeds using a monoclonal antibody to a 16-kDa allergen showed that allergen content of seeds from several transgenic rice plants was markedly lower than that of the seeds from parental wild type rice.

Down regulation of talin alters cell adhesion and the processing of the alpha 5 beta 1 integrin.

The role of talin was addressed by down regulating its expression using an antisense RNA strategy. HeLa cells were transfected with a talin 5' cDNA fragment under the control of the inducible human metallothionein promotor. Isolated clones displayed a decrease in talin level down to 10% of control. The reduction in talin expression dramatically slowed down the kinetics of cell spreading. Mock-transfected cells, spread out onto fibronectin, exhibited large peripheral adhesion plaques. In contrast, cells with reduced talin expression showed smaller focal contacts localized all over the ventral face, and displayed a marked decrease in the number of stress fibers. Immunoprecipitation experiments carried out with a polyclonal antibody on surface-labeled receptor indicated a shift in the mobility for both alpha 5 and beta 1 subunits. Surprisingly, beta 1 integrin chains could not be detected by indirect immunofluorescence using monoclonal antibodies in talin deficient clones. Western blot analysis indicated the presence of two forms of beta 1. We analyzed the processing of beta 1 in normal and talin deficient cells using pulse chase experiments. Normal cells required a minimum of 5 hours for the processing of mature beta 1, while the talin deficient AT22 clone showed that the beta 1 precursor was slowly converted into a very low molecular mass product. Our data demonstrate that talin plays a central role in the establishment of cell-matrix contacts. In addition, down regulation of talin impairs the folding and processing of beta 1 integrins.

Suppression of villin expression by antisense RNA impairs brush border assembly in polarized epithelial intestinal cells.

We have used an antisense RNA strategy to investigate the role of the actin-associated protein, villin, in the brush-border morphogenesis of human intestinal CaCO2 cells. Stable expression of a cDNA encoding antisense villin RNA resulted in the permanent down-regulation of the endogenous villin message and dramatically affected brush-border assembly. Ultrastructural and immunolocalization studies revealed that epithelial cell polarity was largely maintained. However, in contrast to brush-border markers such as dipeptidyl-peptidase IV, the apical localization of sucrase-isomaltase was specifically impaired. Retransfection of the villin antisense-expressing cell line with a cDNA encoding a partial sense villin RNA restored both brush-border assembly and sucrase-isomaltase apical expression. The suggestion that brush-border morphogenesis may be important for the trafficking of certain proteins is discussed.