The pathogenesis of irregular endometrial bleeding, the main reason for stopping contraception with progestins only, is unknown. Based on the recent reappraisal of the mechanisms of menstrual ...bleeding, we hypothesized that matrix metalloproteinases initiate this disorder. Volunteers upon Norplant treatment provided endometrial biopsies at the start of a bleeding episode and during nonbleeding intervals. Focal stromal breakdown, collagen fiber lysis, and collagenase-1 messenger ribonucleic acid were evidenced in most bleeding endometria, but never in the nonbleeding ones. In the breaking down areas, immunolabeling for gelatinase A was strongly increased, and that of progesterone and estrogen receptors was decreased. Explants from bleeding endometria produced high collagenase and gelatinase activities, whereas release from nonbleeding endometria was negligible. Bleeding endometria released more latent and active forms of collagenase-1 and active gelatinases A and B, but less tissue inhibitor of metalloproteinases-1, than nonbleeding endometria. Collagenase-1 release closely correlated with that of interleukin-1alpha. In contrast, N:-acetyl-beta-hexosaminidase and tissue inhibitor of metalloproteinases-2 were similarly released in both groups. Thus, endometrial bleeding occurs together with focal stromal breakdown, collagen lysis, expression and activation of several matrix metalloproteinases, and decreased production of tissue inhibitor of metalloproteinases-1. These results may lead to new pharmacological treatment of this common medical problem.
In the present report the biosynthesis of the integrin alpha-chains endowed with constitutive endoproteolytic cleavage was evaluated in LoVo cells where furin, a subtilisin-like convertase involved ...in post-translational endoproteolytic processing, is not functional. It was found that cell-surface alpha 3, alpha 6 and alpha v subunits were not processed endoproteolytically into heavy and light chains as they were in HT29-D4 cells, a furin-competent cell line. Complete removal of N-linked oligosaccharides and pulse-chase experiments confirmed that the cleavage of the alpha 6 integrin subunit occurring 45 min after translation in HT29 cells did not take place in LoVo cells. Apart from cleavage deficiency, alpha 6 subunit glycosylation, association with beta 4 subunits and targeting to the plasma membrane seemed comparable in LoVo and HT29 cells. The pro-alpha 6 and the pro-alpha 3 subunits immunopurified from LoVo cells were highly sensitive to endoproteolysis by recombinant furin. Furin cleavage was calcium dependent and resulted in the conversion of the 140 kDa pro-alpha 6 into a 120 kDa heavy chain. These results suggest strongly that furin is involved in the endoproteolytic processing of cleavable integrin alpha subunits.
The loss of ABCA1 function leads to Tangier dyslipidemia in humans and to a Tangier-like phenotype in mice, by impairing the transformation of nascent apolipoproteins into mature HDL particles. ...Mechanistically this ensues from the inability of cells to release membrane lipids and cholesterol. Whereas the ability of ABCA1 to promote phospholipid effluxes, surface binding of apolipoproteins and outward flip of membrane lipids has been documented, the relationship between this series of ABCA1-dependent events is still elusive.Here we provide evidence that i) lipid effluxes require both flip of membrane lipids and binding of apolipoproteins to the cell surface, ii) apolipoprotein A-I binding depends on structural determinants on ABCA1, and iii) phospholipid effluxes can be modulated by engineered mutations on the structural determinants identified on ABCA1.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The integrin alpha6beta1 and its main ligand laminin-111 are overexpressed in glioblastoma, as compared with normal brain tissue, suggesting they may be involved in glioblastoma malignancy. To ...address this question, we stably expressed the alpha6 integrin subunit in the U87 cell line via retroviral-mediated gene transfer. We show that cell surface expression of the alpha6beta1 integrin led to dramatic changes in tumor U87 cell behavior, both in vitro and in vivo. Nude mice receiving either subcutaneous or intracerebral inoculation of alpha6beta1-expressing cells developed substantially more voluminous tumors than mice injected with control cells. The difference in tumor growth was associated with a marked increase in vascularization in response to alpha6beta1 integrin expression and may also be related to changes in the balance between cell proliferation and survival. Indeed, expression of alpha6beta1 enhanced proliferation and decreased apoptosis of U87 cells both in the tumor and in vitro. Additionally, we demonstrate that alpha6beta1 is implicated in glioblastoma cell migration and invasion and that laminin-111 might mediate dissemination of alpha6beta1-positive cells in vivo. Our results highlight for the first time the considerable role of the integrin alpha6beta1 in glioma progression.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Crosstalk between integrins is involved in the regulation of various cell functions including cell migration. Here we identify the interplay between the integrins alphavbeta5/beta6 and alpha2beta1 ...during cell migration toward type I collagen. Human colon cancer cell lines HT29-D4 and SW480 were used as cell models. To improve our understanding of the consequences of alphavbeta5/beta6 function on alpha2beta1, we decreased the expression of alphav integrins by either siRNA or lysosomal targeting strategies or inhibited their function using, as antagonists, blocking antibodies or disintegrins. In all cases, we observed a greatly enhanced alpha2beta1 integrin-dependent cell migration associated with focal adhesion rearrangements and increased outside-in signaling as demonstrated by elevated phosphorylation of focal adhesion kinase and MAPKinase (ERK1 and ERK2). The alphavbeta5/beta6-dependent limitation of alpha2beta1 function could be overridden by TS2/16, an activating anti-beta1 antibody. Interestingly, compared to control cells, the pharmacological inhibition of PI3Kinase or the siRNA-mediated knockdown of AKT had little effect on the high alpha2beta1-mediated cell migration observed in the absence of alphav integrins or following activation of alpha2beta1 integrins by the TS2/16. These results suggest that integrins alphavbeta5/beta6 repress alpha2beta1 possibly by interfering with their activation process and thereby modify the cell signaling regulation of alpha2beta1-mediated migration.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Background information. Previous studies have reported that cross-talk between integrins may be an important regulator of integrin-ligand binding and subsequent signalling events that control a ...variety of cell functions in many tissues. We previously demonstrated that alpha v beta 5/beta 6 integrin represses alpha 2 beta 1-dependent cell migration. The alpha v subunits undergo an endoproteolytic cleavage by protein convertases, whose role in tumoral invasion has remained controversial. Results. Inhibition of convertases by the convertase inhibitor alpha 1-PDX (alpha 1-antitrypsin Portland variant), leading to the cell-surface expression of an uncleaved form of the av integrin, stimulated cell migration toward type I collagen. Under convertase inhibition, alpha 2 beta 1 engagement led to enhanced phosphorylation of both FAK (focal adhesion kinase) and MAPK (mitogen-activated protein kinase). This outside-in signalling stimulation was associated with increased levels of activated integrin located in larger than usual focal-adhesion structures and a cell migration that was independent of the PI3K (phosphoinositide 3-kinase)/Akt (also called protein kinase B) pathway. Conclusions. The increase in cell migration observed upon convertases inhibition appears to be due to the up-regulation of beta 1 integrins and to their location in larger focal-adhesion structures. The endoproteolytic cleavage of av subunits is necessary for alpha v beta 5/beta 6 integrin to control alpha 2 beta 1 function and could thus play an essential role in colon cancer cell migration.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Most matrix metalloproteinases (MMPs) are secreted as inactive proenzymes. Their expression is well documented in several human tissues, but their activators in vivo are still unknown. To address ...this question, the activation of progelatinase B (proMMP-9) in the human endometrium was selected as a model system. ProMMP-9 was detected by gelatin zymography in homogenates of fresh endometrial tissue sampled during all phases of the menstrual cycle, whereas its active form was observed only during the late secretory and menstrual phases. Furthermore, proMMP-9 was expressed and activated in endometrial explants sampled outside the perimenstrual phase and cultured in the absence of both progesterone and oestradiol, mimicking the menstrual condition in vivo. Analysis of such tissue cultures by gelatin zymography and Western blotting showed that activation of proMMP-9 depended on a secreted factor and was selectively inhibited by either a synthetic inhibitor of stromelysin 1 (MMP-3) or a monoclonal antibody that specifically blocks MMP-3, thus providing strong evidence for the activation of proMMP-9 in vivo by MMP-3. The activation of proMMP-3 was itself inhibited by a broad-range MMP inhibitor in most cultures, but seemed to involve multiple pathways, implying both serine proteinases and metalloproteinases, which could operate in parallel or sequentially.
Some integrin α subunits undergo a post-translational cleavage in their extracellular domain. However, the role of this cleavage in integrin function is unclear. Enzymes involved in this maturation ...belong to the subtilisin-like endoprotease family (convertases). To understand the role of the α subunit cleavage in integrin function, we have designed stable transfectants (PDX39P cells) expressing α1-PDX, a convertase inhibitor. Immunoprecipitation of cell surface proteins from PDX39P showed that α3, α6 and αvintegrins lack endoproteolytic cleavage. We have compared adhesion between PDX39P cells and mock-transfected cells on different extracellular matrix proteins. No difference in adhesion could be observed on laminin-1 and type I collagen, while attachment of PDX39P cells to vitronectin (ligand of the αvβ5integrin) was dramatically reduced. The reduced adhesion of PDX39P cells was not due to changes in integrin affinity as determined by solid-phase receptor assay in a cell-free environment. Intracellular signaling pathways activated by αv integrin ligation were also affected in PDX39P cells. It thus seems that the absence of endoproteolytic cleavage of αv integrins has important consequences on signal transduction pathways leading to alterations in integrin function such as cell adhesion.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The pathogenesis of irregular endometrial bleeding, the main reason for
stopping contraception with progestins only, is unknown. Based on the
recent reappraisal of the mechanisms of menstrual ...bleeding, we
hypothesized that matrix metalloproteinases initiate this disorder.
Volunteers upon Norplant treatment provided endometrial biopsies at the
start of a bleeding episode and during nonbleeding intervals. Focal
stromal breakdown, collagen fiber lysis, and collagenase-1
messenger ribonucleic acid were evidenced in most bleeding
endometria, but never in the nonbleeding ones. In the breaking down
areas, immunolabeling for gelatinase A was strongly increased, and that
of progesterone and estrogen receptors was decreased. Explants from
bleeding endometria produced high collagenase and gelatinase
activities, whereas release from nonbleeding endometria was negligible.
Bleeding endometria released more latent and active forms of
collagenase-1 and active gelatinases A and B, but less tissue inhibitor
of metalloproteinases-1, than nonbleeding endometria. Collagenase-1
release closely correlated with that of interleukin-1α. In contrast,
N-acetyl-β-hexosaminidase and tissue inhibitor of
metalloproteinases-2 were similarly released in both groups. Thus,
endometrial bleeding occurs together with focal stromal breakdown,
collagen lysis, expression and activation of several matrix
metalloproteinases, and decreased production of tissue inhibitor of
metalloproteinases-1. These results may lead to new pharmacological
treatment of this common medical problem.
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