FASTKD2 is associated with memory and hippocampal structure in older adults.

Memory impairment is the cardinal early feature of Alzheimer's disease, a highly prevalent disorder whose causes remain only partially understood. To identify novel genetic predictors, we used an integrative genomics approach to perform the largest study to date of human memory (n=14 781). Using a genome-wide screen, we discovered a novel association of a polymorphism in the pro-apoptotic gene FASTKD2 (fas-activated serine/threonine kinase domains 2; rs7594645-G) with better memory performance and replicated this finding in independent samples. Consistent with a neuroprotective effect, rs7594645-G carriers exhibited increased hippocampal volume and gray matter density and decreased cerebrospinal fluid levels of apoptotic mediators. The MTOR (mechanistic target of rapamycin) gene and pathways related to endocytosis, cholinergic neurotransmission, epidermal growth factor receptor signaling and immune regulation, among others, also displayed association with memory. These findings nominate FASTKD2 as a target for modulating neurodegeneration and suggest potential mechanisms for therapies to combat memory loss in normal cognitive aging and dementia.

Anxiety is related to Alzheimer cerebrospinal fluid markers in subjects with mild cognitive impairment.

BACKGROUND: Anxiety, apathy and depression are common in subjects with mild cognitive impairment (MCI) and may herald Alzheimer's disease (AD). We investigated whether these symptoms correlated with cerebrospinal fluid (CSF) markers for AD in subjects with MCI. Method Subjects with MCI (n=268) were selected from the 'Development of screening guidelines and criteria for pre-dementia Alzheimer's disease' (DESCRIPA) and Alzheimer's Disease Neuroimaging Initiative (ADNI) studies. We measured amyloid ß(1-42) protein (Aß42) and total tau (t-tau) in CSF. Neuropsychiatric symptoms were measured with the Neuropsychiatric Inventory. RESULTS: Depressive symptoms were reported by 55 subjects (21%), anxiety by 35 subjects (13%) and apathy by 49 subjects (18%). The presence of anxiety was associated with abnormal CSF Aß42 [odds ratio (OR) 2.3, 95% confidence interval (CI) 1.6-3.3] and t-tau (OR 2.6, 95% CI 1.9-3.6) concentrations and with the combination of abnormal concentrations of both Aß42 and t-tau (OR 3.1, 95% CI 2.0-4.7). The presence of agitation and irritability was associated with abnormal concentrations of Aß42 (agitation: OR 1.6, 95% CI 1.1-2.3; irritability: OR 2.2, 95% CI 1.5-3.3). Symptoms of depression and apathy were not related to any of the CSF markers. CONCLUSIONS: In subjects with MCI, symptoms of anxiety, agitation and irritability may reflect underlying AD pathology, whereas symptoms of depression and apathy do not.

The alpha 6 beta 4 integrin and epithelial cell migration.

Although the involvement of alpha 6 beta 4, an integrin laminin receptor, in hemidesmosome organization has dominated the study of this integrin, recent studies are revealing novel functions for alpha 6 beta 4 in the migration of epithelial and carcinoma cells. The engagement of laminin by alpha 6 beta 4 can stabilize actin-rich protrusions and mediate traction forces necessary for cell movement. This integrin also has a significant impact on signaling molecules that stimulate migration and invasion, especially PI3-K and Rho GTPases. Activation of PI3-K by alpha 6 beta 4 enhances the formation of actin protrusions, and it may stimulate the function of other integrins, such as alpha 3 beta 1, that are also important for epithelial migration. Signaling through alpha 6 beta 4 may not always depend on the adhesive functions of this integrin, a possibility that has profound implications for migration and invasion because it implies that the ability of alpha 6 beta 4 to stimulate these processes is not limited to specific matrix environments.

Postoperative pulmonary complications following major head and neck cancer surgery.

The primary aim of this observational study was to describe the incidence of postoperative pulmonary complications (PPCs) in 60 consecutive, surgically treated head and neck cancer patients requiring free flap reconstruction and tracheostomy, using both a prospective and a retrospective outcome measure. Secondary aims were to identify risk factors for PPC development, explore the effects of PPC on outcomes, and describe the provision of postoperative physiotherapy in this population. Postoperative pulmonary complications occurred in nine (15%) patients based on the Melbourne Group Scale and 27 (45%) patients based on Health Information Service coding data. The occurrence of a PPC was not statistically correlated with age, smoking history, comorbidities, operative time, or type of resection or free flap. Patients who developed a PPC, compared to those who did not, had a higher preoperative body mass index (P=0.022) and were more likely to be sat out of bed earlier post-surgery (P=0.038). Overall, patients required a median of 9.0 (interquartile range 7.0-11.0) physiotherapy sessions. Patients developing a PPC required significantly more physiotherapy sessions (P=0.007) and additional days of supplemental oxygen (P=0.022) as compared to those without a PPC, despite a similar hospital length of stay. In future, targeted physiotherapy interventions may reduce PPCs in this population.

Interferon gamma and lipopolysaccharide promote macrophage adherence to basement membrane glycoproteins.

The ability of thioglycollate (TG)-elicited peritoneal macrophages, a population of recently recruited monocytes, to adhere to the basement membrane glycoproteins laminin and type IV collagen is not a constitutive function of these cells. Adherence can be induced, however, by treatment with IFN-gamma and LPS. In general, IFN-gamma is more potent than LPS in promoting this adherence. Maximal adherence, however, is observed when IFN-gamma (greater than or equal to 5 U/ml) is used together with LPS (2.0 ng/ml). These requirements parallel the conditions needed to obtain tumoricidal activation of TG-elicited macrophages. Adherence to laminin, in the presence of these stimuli, is transient, being maximal at 8 h after their addition and diminishing with longer periods of incubation. In contrast, adherence to type IV collagen does not appear to be transient and IFN-gamma and LPS induce a more prolonged association of macrophages with this substratum.

MRI of hippocampal volume loss in early Alzheimer's disease in relation to ApoE genotype and biomarkers.

Hippocampal volume change over time, measured with MRI, has huge potential as a marker for Alzheimer's disease. The objectives of this study were: (i) to test if constant and accelerated hippocampal loss can be detected in Alzheimer's disease, mild cognitive impairment and normal ageing over short periods, e.g. 6-12 months, with MRI in the large multicentre setting of the Alzheimer's Disease Neuroimaging Initiative (ADNI); (ii) to determine the extent to which the polymorphism of the apolipoprotein E (ApoE) gene modulates hippocampal change; and (iii) to determine if rates of hippocampal loss correlate with cerebrospinal fluid (CSF) biomarkers of Alzheimer's disease, such as the beta-amyloid (Abeta(1-42)) and tau proteins (tau). The MRI multicentre study included 112 cognitive normal elderly individuals, 226 mild cognitive impairment and 96 Alzheimer's disease patients who all had at least three successive MRI scans, involving 47 different imaging centres. The mild cognitive impairment and Alzheimer's disease groups showed hippocampal volume loss over 6 months and accelerated loss over 1 year. Moreover, increased rates of hippocampal loss were associated with presence of the ApoE allele epsilon4 gene in Alzheimer's disease and lower CSF Abeta(1-42) in mild cognitive impairment, irrespective of ApoE genotype, whereas relations with tau were only trends. The power to measure hippocampal change was improved by exploiting correlations statistically between successive MRI observations. The demonstration of considerable hippocampal loss in mild cognitive impairment and Alzheimer's disease patients over only 6 months and accelerated loss over 12 months illustrates the power of MRI to track morphological brain changes over time in a large multisite setting. Furthermore, the relations between faster hippocampal loss in the presence of ApoE allele epsilon4 and decreased CSF Abeta(1-42) supports the concept that increased hippocampal loss is an indicator of Alzheimer's disease pathology and a potential marker for the efficacy of therapeutic interventions in Alzheimer's disease.

Fixed- or controlled-dose mycophenolate mofetil with standard- or reduced-dose calcineurin inhibitors: the Opticept trial.

Mycophenolate mofetil (MMF) was developed with cyclosporine as a fixed-dose immunosuppressant. More recent data indicate a relationship between mycophenolic acid (MPA) exposure in individuals and clinical endpoints of rejection and toxicity. This 2-year, open-label, randomized, multicenter trial compared the efficacy and safety of concentration-controlled MMF (MMF(CC)) dosing with a fixed-dose regimen in 720 kidney recipients. Patients received either (A) MMF(CC) and reduced-level calcineurin inhibitor (MMF(CC)/CNI(RL)); (B) MMF(CC) and standard-level CNI (MMF(CC)/CNI(SL)); or (C) fixed-dose MMF and CNI(SL) (MMF(FD)/CNI(SL)). Antibody induction and steroid use were according to center practice. The primary endpoint was noninferiority (alpha= 0.05) of group A versus group C for treatment failure (including biopsy-proven acute rejection [BPAR], graft loss and death) at 1 year. Although mean CNI trough levels in group A did not reach the prespecified targets, they were statistically lower than those in groups B and C (p < or = 0.01 for each comparison). BPAR rates (8.5%) were low across groups. Group A had 19% fewer treatment failures (23% vs. 28%, p = 0.18). MMF doses were highest (p < 0.05), with withdrawals for adverse events the fewest (p = 0.02), in group A. Of the 80% of subjects taking tacrolimus (Tac), those with higher MPA exposure had significantly less rejection (p < 0.001) and diarrhea correlated with Tac, but not with MPA levels. Thus, MMF(CC) with low-dose CNI resulted in outcomes not inferior to those with standard CNI exposure and MMF(FD), indicating potential utility of MMF(CC) in CNI-sparing regimens.

Regulation of alpha 6 beta 1 integrin laminin receptor function by the cytoplasmic domain of the alpha 6 subunit.

The alpha 6 beta 1 integrin is expressed on the macrophage surface in an inactive state and requires cellular activation with PMA or cytokines to function as a laminin receptor (Shaw, L. M., J. M. Messier, and A. M. Mercurio. 1990. J. Cell Biol. 110:2167-2174). In the present study, the role of the alpha 6 subunit cytoplasmic domain in alpha 6 beta 1 integrin activation was examined. The use of P388D1 cells, an alpha 6-integrin deficient macrophage cell line, facilitated this analysis because expression of either the alpha 6A or alpha 6B subunit cDNAs restores their activation responsive laminin adhesion (Shaw, L. S., M. Lotz, and A. M. Mercurio. 1993. J. Biol. Chem. 268:11401-11408). A truncated alpha 6 cDNA, alpha 6-delta CYT, was constructed in which the human cytoplasmic domain sequence was deleted after the GFFKR pentapeptide. Expression of this cDNA in P388D1 cells resulted in the surface expression of a chimeric alpha 6-delta CYT beta 1 integrin that was unable to mediate laminin adhesion or increase this adhesion in response to PMA under normal conditions, i.e., in medium that contained physiological concentrations of Ca++ and Mg++. The alpha 6A-delta CYT transfectants adhered to laminin, however, when Ca++/Mg++ was replaced with 150 microM Mn++. We also assessed the role of serine phosphorylation in the regulation of alpha 6A beta 1 integrin function by site-directed mutagenesis of the two serine residues present in the alpha 6A cytoplasmic domain because this domain is phosphorylated on serine residues in response to stimuli that activate the laminin receptor function of alpha 6 A beta 1. Point mutations were introduced in the alpha 6A cDNA that changed either serine residue #1064 (M1) or serine residue #1071 (M2) to alanine residues. In addition, a double mutant (M3) was constructed in which both serine residues were changed to alanine residues. P388D1 transfectants which expressed these serine mutations adhered to laminin in response to PMA to the same extent as cells transfected with wild-type alpha 6A cDNA. These findings provide evidence for a novel mode of integrin regulation that is distinct from that reported for other regulated integrins (O'Toole, T. E., D. Mandelman, J. Forsyth, S. J. Shattil, E. F. Plow, and M. H. Ginsberg. 1991. Science (Wash. DC). 254:845-847. Hibbs, M. L., H. Xu, S. A. Stacker, and T. A. Springer. 1991. Science (Wash. DC). 251:1611-1613), and they demonstrate that serine phosphorylation of the alpha 6A cytoplasmic domain is not involved in this regulation.

Macrophage interactions with laminin: PMA selectively induces the adherence and spreading of mouse macrophages on a laminin substratum.

The ability of thioglycollate (TG)-elicited mouse peritoneal macrophages to adhere to a laminin substratum has been studied. These cells do not adhere to laminin-coated (20 micrograms/ml) surfaces, but the addition of phorbol myristate acetate (PMA; 50 ng/ml) results in their rapid adherence and spreading on this substratum. TG-elicited and PMA-activated macrophages, however, can bind soluble laminin. Macrophages adhere to fibronectin-coated surfaces and tissue culture plastic without PMA stimulation, and PMA does not increase the number of cells that adhere to these surfaces. The predominant surface proteins that bind specifically to laminin-Sepharose exhibit an Mr of 67 and 36 kD, but the expression of these proteins does not increase after PMA stimulation. Laminin receptor antibodies immunoprecipitate the 67-kD protein from radiolabled surface lysates and are capable of blocking macrophage adherence to a laminin substratum. Indirect immunofluorescence microscopy indicates that PMA stimulation does not increase receptor expression, but that it may induce the aggregation of the receptor on the cell surface. PMA stimulation also promotes macrophage spreading and induces a reorganization of the actin cytoskeleton. Taken together, these data indicate the mechanism by which PMA promotes macrophage adherence to laminin does not involve increased 67-kD receptor surface expression, but that it is related to the changes in cytoskeletal and receptor surface organization that occur in response to PMA stimulation.

Release of cAMP gating by the alpha6beta4 integrin stimulates lamellae formation and the chemotactic migration of invasive carcinoma cells.

The alpha6beta4 integrin promotes carcinoma in-vasion by its activation of a phosphoinositide 3-OH (PI3-K) signaling pathway (Shaw, L.M., I. Rabinovitz, H.H.-F. Wang, A. Toker, and A.M. Mercurio. Cell. 91: 949-960). We demonstrate here using MDA-MB-435 breast carcinoma cells that alpha6beta4 stimulates chemotactic migration, a key component of invasion, but that it has no influence on haptotaxis. Stimulation of chemotaxis by alpha6beta4 expression was observed in response to either lysophosphatidic acid (LPA) or fibroblast conditioned medium. Moreover, the LPA-dependent formation of lamellae in these cells is dependent upon alpha6beta4 expression. Both lamellae formation and chemotactic migration are inhibited or "gated" by cAMP and our results reveal that a critical function of alpha6beta4 is to suppress the intracellular cAMP concentration by increasing the activity of a rolipram-sensitive, cAMP-specific phosphodiesterase (PDE). This PDE activity is essential for lamellae formation, chemotactic migration and invasion based on data obtained with PDE inhibitors. Although PI3-K and cAMP-specific PDE activities are both required to promote lamellae formation and chemotactic migration, our data indicate that they are components of distinct signaling pathways. The essence of our findings is that alpha6beta4 stimulates the chemotactic migration of carcinoma cells through its ability to influence key signaling events that underlie this critical component of carcinoma invasion.

The activation dependent adhesion of macrophages to laminin involves cytoskeletal anchoring and phosphorylation of the alpha 6 beta 1 integrin.

Macrophages require activation with either PMA (Mercurio, A. M., and L. M. Shaw. 1988. J. Cell Biol. 107:1873-1880) or interferon-gamma (Shaw, L. M., and A. M. Mercurio. 1989. J. Exp. Med. 169:303-308) to adhere to a laminin substratum. In the present study, we identified an integrin laminin receptor on macrophages and characterized cellular changes that occur in response to PMA activation that facilitate laminin adhesion. A monoclonal antibody (GoH3) that recognizes the integrin alpha 6 subunit (Sonnenberg, A., H. Janssen, F. Hogervorst, J. Calafat, and J. Hilgers. 1987. J. Biol. Chem. 262:10376-10383) specifically inhibited adhesion to laminin-coated surfaces. This antibody precipitated an alpha 6 beta 1 heterodimer (Mr 130/110 kD) from 125I surface-labeled macrophages. The amount of radiolabeled receptor on the cell surface did not increase after PMA activation. Thus, the induction of laminin adhesion cannot be attributed to de novo or increased surface expression of alpha 6 beta 1. By initially removing the Triton X-100-soluble fraction of macrophages and then disrupting the remaining cytoskeletal framework, we observed that 75% of the alpha 6 beta 1 heterodimer on the cell surface is anchored to the cytoskeleton in macrophages that had adhered to a laminin substratum in response to PMA. Significant cytoskeletal anchoring of this receptor was not observed in macrophages that had adhered to fibronectin or tissue culture plastic, nor was it seen in nonadherent cells. PMA also induced phosphorylation of the cytoplasmic domain of the alpha 6 subunit, but not the beta 1 subunit. Phosphorylated alpha 6 was localized to the cytoskeletal fraction only in macrophages plated on a laminin substratum. In summary, our results support a mechanism for the regulation of macrophage adhesion to laminin that involves specific and dynamic matrix integrin-cytoskeletal interactions that may be facilitated by integrin phosphorylation.

Evidence that distinct states of the integrin alpha6beta1 interact with laminin and an ADAM.

Integrins can exist in different functional states with low or high binding capacity for particular ligands. We previously provided evidence that the integrin alpha6beta1, on mouse eggs and on alpha6-transfected cells, interacted with the disintegrin domain of the sperm surface protein ADAM 2 (fertilin beta). In the present study we tested the hypothesis that different states of alpha6beta1 interact with fertilin and laminin, an extracellular matrix ligand for alpha6beta1. Using alpha6-transfected cells we found that treatments (e.g., with phorbol myristate acetate or MnCl2) that increased adhesion to laminin inhibited sperm binding. Conversely, treatments that inhibited laminin adhesion increased sperm binding. Next, we compared the ability of fluorescent beads coated with either fertilin beta or with the laminin E8 fragment to bind to eggs. In Ca2+-containing media, fertilin beta beads bound to eggs via an interaction mediated by the disintegrin loop of fertilin beta and by the alpha6 integrin subunit. In Ca2+-containing media, laminin E8 beads did not bind to eggs. Treatment of eggs with phorbol myristate acetate or with the actin disrupting agent, latrunculin A, inhibited fertilin bead binding, but did not induce laminin E8 bead binding. Treatment of eggs with Mn2+ dramatically increased laminin E8 bead binding, and inhibited fertilin bead binding. Our results provide the first evidence that different states of an integrin (alpha6beta1) can interact with an extracellular matrix ligand (laminin) or a membrane-anchored cell surface ligand (ADAM 2).

Pharmacokinetics and bioequivalence of 20 mg omeprazole capsule in 24 healthy Korean male volunteers.

OBJECTIVE: A randomized, two-way, crossover bioequivalence study in 24 healthy Korean male volunteers was conducted to compare bioequivalence of two brands of 20 mg omeprazole capsules, Hutex omeprazole (Hutex Pharm Co. Korea) as a test and Yuhan Losec (Yuhan Co. Ltd., Korea) as a reference drug. VOLUNTEERS AND METHODS: Subjects were administered single dosage of 1 capsule of 20 mg of each formulation with 240 ml of water after 10 hs overnight fasting on 2 treatment days separated by one-week washout period. After dosing, serial blood sampling was held during 9 hs. Plasma was analyzed for omeprazole by a validated HPLC method with ultraviolet detection in the range of 10 approximately 1,000 ng/ml with the lowest limit of quantification of 10 ng/ml. RESULTS: Several pharmacokinetic (PK) parameters were determined from the plasma samples, and data from reference and test formulations in the plasma were represented such as AUC0-t (1,223.3 vs 1,284.3 ng x h/ml), [formula in text](1,311.1 vs 1,410.0 ng x h/ml), Cmax (598.7 vs 598.1 ng/ml), tmax (1.9 vs 1.9 h), t1/2 (1.3 vs 1.4 h) and Ke (0.67 vs 0.67 h-1), respectively. AUC0-t, [formula in text] and Cmax were tested for bioequivalence after log-transformation of plasma data. PK parameters with 90% confidence interval (CI) of test/reference ratio based on ANOVA analysis were 0.961 approximately 1.135 for AUC0-t, 0.968 approximately 1.144 for [formula in text] and 0.951 approximately 1.117 for Cmax. CONCLUSIONS: PK parameters with 90% CI were within the bioequivalence range of 80 - 125% of FDA statistical limit. Therefore, both omeprazole formulations were bioequivalent during fasting state in these healthy Korean male volunteers.

FACIT collagens: diverse molecular bridges in extracellular matrices.

The collagens form a large family of proteins. Collagen fibrils, composed of staggered arrays of fibrillar collagen molecules (types I, II, III, V and XI), provide a supporting scaffold for extracellular matrices of connective tissues. The non-fibrillar collagens are less abundant than the fibrillar collagens, but it is becoming clear that they have important functions in the matrix. Recently, a group with unique structural characteristics has been defined and named the FACIT (Fibril-Associated Collagens with Interrupted Triple-helices) group. There is evidence that these collagens may serve as molecular bridges that are important for the organization and stability of extracellular matrices.

Polycystic ovary syndrome: a transgenerational evolutionary adaptation.

Polycystic ovary syndrome has a common association with anovulatory infertility, while the physical symptoms are often associated with the increased androgens that are part of the endocrine profile. There is a well-recognised association with lipid and glucose metabolism anomalies and, when undergoing ovulation induction, ovarian hyperstimulation syndrome. This common condition is familial, but a contributory gene has yet to be found. The question of why a gene that predisposes to anovulation, diabetes and heart disease might have perpetuated so frequently is addressed. Three hypotheses for evolutionary advantage are discussed. The food deprivation hypothesis considers the role of the observed increase in ovulation when women with the condition lose weight in relation to seasonality. The refeeding hypothesis considers the androgenic and slightly enhanced anabolic metabolism in relation to periods of privation and the advantage of preferential early ovulation when refeeding after a period of privation. The transgenerational privation hypothesis considers the effect of persistent, severe, yet subfatal privation on individuals both in utero and throughout life. While an androgenic, anabolic state would improve efficiency in the use of food for protein synthesis and fat storage, benefiting the fetus both in relation to its in utero development and neonatal survival, survival and reproductive capacity as an adult benefits by a genotype expressing itself in women of successive generations.

Removal of gadolinium by peritoneal dialysis.

An association between gadolinium-containing contrast and the development of nephrogenic systemic fibrosis (NSF) has been increasingly recognized. For patients receiving hemodialysis (HD) who are exposed to gadolinium, the Federal Drug Administration (FDA) recommends HD to remove this contrast agent in order to minimize the risk of NSF. This study examines if gadolinium can be removed by frequent exchanges by peritoneal dialysis (PD). Following administration of 0.1 mmol/kg of gadodiamide to a patient with end-stage renal disease, the serum clearance of this contrast agent by automated PD was examined. 10 and 15 exchanges of PD using an automated cycler were respectively performed during the first and second 24-hour periods after gadolinium exposure. Serum gadolinium levels were measured 1 hour after the gadolinium administration, then at 24 and 48 hours after PD was initiated. 90% of the gadolinium was removed from the circulation in 2 days with a regimen of 10-15 exchanges per day of PD. For patients on chronic maintenance PD who receive gadolinium, our case suggests that a temporary intensive automated PD regimen, aimed at maximizing clearance of this contrast agent immediately after exposure, could be an effective alternative when institution of HD is problematic.

Identification of insulin receptor substrate 1 (IRS-1) and IRS-2 as signaling intermediates in the alpha6beta4 integrin-dependent activation of phosphoinositide 3-OH kinase and promotion of invasion.

Expression of the alpha6beta4 integrin increases the invasive potential of carcinoma cells by a mechanism that involves activation of phosphoinositide 3-OH kinase (PI3K). In the present study, we investigated the signaling pathway by which the alpha6beta4 integrin activates PI3K. Neither the alpha6 nor the beta4 cytoplasmic domain contains the consensus binding motif for PI3K, pYMXM, indicating that additional proteins are likely to be involved in the activation of this lipid kinase by the alpha6beta4 integrin. We identified insulin receptor substrate 1 (IRS-1) and IRS-2 as signaling intermediates in the activation of PI3K by the alpha6beta4 integrin. IRS-1 and IRS-2 are cytoplasmic adapter proteins that do not contain intrinsic kinase activity but rather function by recruiting proteins to surface receptors, where they organize signaling complexes. Ligation of the alpha6beta4 receptor promotes tyrosine phosphorylation of IRS-1 and IRS-2 and increases their association with PI3K, as determined by coimmunoprecipitation. Moreover, we identified a tyrosine residue in the cytoplasmic domain of the beta4 subunit, Y1494, that is required for alpha6beta4-dependent phosphorylation of IRS-2 and activation of PI3K in response to receptor ligation. Most importantly, Y1494 is essential for the ability of the alpha6beta4 integrin to promote carcinoma invasion. Taken together, these results imply a key role for the IRS proteins in the alpha6beta4-dependent promotion of carcinoma invasion.

Regulation of cellular interactions with laminin by integrin cytoplasmic domains: the A and B structural variants of the alpha 6 beta 1 integrin differentially modulate the adhesive strength, morphology, and migration of macrophages.

Several integrin alpha subunits have structural variants that are identical in their extracellular and transmembrane domains but that differ in their cytoplasmic domains. The functional significance of these variants, however, is unknown. In the present study, we examined the possibility that the A and B variants of the alpha 6 beta 1 integrin laminin receptor differ in function. For this purpose, we expressed the alpha 6A and alpha 6B cDNAs, as well as a truncated alpha 6 cDNA (alpha 6-delta CYT) in which the cytoplasmic domain sequence was deleted after the GFFKR pentapeptide, in P388D1 cells, an alpha 6 deficient macrophage cell line. Populations of stable alpha 6A, alpha 6B, and alpha 6-delta CYT transfectants that expressed equivalent levels of cell surface alpha 6 were obtained by fluorescence-activated cell sorter and shown to form heterodimers with endogenous beta 1 subunits. Upon attachment to laminin, the alpha 6A transfectants extended numerous pseudopodia. In contrast, the alpha 6B transfectants remained rounded and extended few processes. The transfectants were also examined for their ability to migrate toward a laminin substratum using Transwell chambers. The alpha 6A transfectants were three- to fourfold more migratory than the alpha 6B transfectants. The alpha 6-delta CYT transfectants did not attach to laminin in normal culture medium, but they did attach in the presence of Mn2+. The alpha 6-delta CYT transfectants migrated to a lesser extent than either the alpha 6A or alpha 6B transfectants in the presence of Mn2+. The alpha 6 transfectants differed significantly in the concentration of substratum bound laminin required for half-maximal adhesion in the presence of Mn2+:alpha 6A (2.1 micrograms/ml), alpha 6B (6.3 micrograms/ml), and alpha 6-delta CYT (8.8 micrograms/ml). Divalent cation titration studies revealed that these transfectants also differed significantly in both the [Ca2+] and [Mn2+] required to obtain half-maximal adhesion to laminin. These data demonstrate that the A and B variants of the alpha 6 cytoplasmic domain can differentially modulate the function of the alpha 6 beta 1 extracellular domain.

Function of the integrin alpha 6 beta 1 in metastatic breast carcinoma cells assessed by expression of a dominant-negative receptor.

The involvement of the alpha 6 beta a integrin, a laminin receptor, in breast carcinoma progression needs to be addressed rigorously. We report that a human breast carcinoma cell line, MDA-MB-435, known to be highly invasive and metastatic, expresses three potential integrin laminin receptors: alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 1, but uses only alpha 6 beta 1 to mediate adhesion and migration on laminin matrices. To investigate the contribution of alpha 6 beta 1 to the aggressive behavior of these cells, we developed a dominant-negative strategy for knocking out alpha 6 beta 1 function that involved expression of a cytoplasmic domain deletion mutant of the beta 4 integrin subunit by cDNA transfection. Stable transfectants of MDA-MB-435 cells that expressed this mutant beta 4 subunit were inhibited dramatically in their ability to adhere and migrate on laminin matrices, and their capacity to invade Matrigel was reduced significantly. These findings support the hypothesis that alpha 6 beta 1 is important for breast cancer progression. Moreover, this approach is a powerful method that should be useful in assessing the role of alpha 6 beta 1 in other cells.

Drg-1 as a differentiation-related, putative metastatic suppressor gene in human colon cancer.

A gene related to cell differentiation was identified by differential display as a candidate suppressor of metastases in colon cancer. This gene, with a full-length cDNA of 3 kb, is expressed in normal colon and primary colon cancer tissues and cell lines but not in their metastatic counterparts. A GenBank search found that it is identical to a recently cloned gene, differentiation-related gene-1 (Drg-1), isolated from differentiated HT-29 colon cancer cells. Stable transfection of the SW620 metastatic colon cancer cell line with Drg-1 cDNA induced morphological changes consistent with differentiation and up-regulated the expression of several colonic epithelial cell differentiation markers (alkaline phosphatase, carcinoembryonic antigen, and E-cadherin). Moreover, the expression of Drg-1 is controlled by several known cell differentiation reagents, such as ligands of peroxisome proliferator-activated receptor gamma (troglitazone and BRL46593) and of retinoid X receptor (LG268), and histone deacetylase inhibitors (trichostatin A, suberoylanilide hydroxamic acid, and tributyrin). A synergistic induction of Drg-1 expression was seen with the combination of tributyrin and a low dose of 5'-aza-2'-dexoycytidine (100 nM), an inhibitor of DNA methylation. Functional studies revealed that overexpression of Drg-1 in metastatic colon cancer cells reduced in vitro invasion through Matrigel and suppressed in vivo liver metastases in nude mice. We propose that Drg-1 suppresses colon cancer metastasis by inducing colon cancer cell differentiation and partially reversing the metastatic phenotype.