Vitamin A Status Modulates Epithelial Mesenchymal Transition in the Lung: The Role of Furin.

Vitamin A deficiency (VAD) induced TGF-ß hyperactivation and reduced expression of cell adhesion proteins in the lung, suggesting that the disruption of retinoic acid (RA) signaling leads to epithelial-mesenchymal transition (EMT). To elucidate the role of lung vitamin A status in EMT, several EMT markers and the expression of the proprotein convertase furin, which activates TGF-ß, were analyzed in two experimental models. Our in vivo model included control rats, VAD rats, and both control rats and VAD rats, treated with RA. For the in vitro studies, human bronchoalveolar epithelial cells treated with RA were used. Our data show that EMT and furin are induced in VAD rats. Furthermore, furin expression continues to increase much more markedly after treatment of VAD rats with RA. In control rats and cell lines, an acute RA treatment induced a significant increase in furin expression, concomitant with changes in EMT markers. A ChIP assay demonstrated that RA directly regulates furin transcription. These results emphasize the importance of maintaining vitamin A levels within the physiological range since both levels below and above this range can cause adverse effects that, paradoxically, could be similar. The role of furin in EMT is discussed.

In vitro assessment of the photo(geno)toxicity associated with Lapatinib, a Tyrosine Kinase inhibitor.

The epidermal growth factor receptors EGFR and HER2 are the main targets for tyrosine kinase inhibitors (TKIs). The quinazoline derivative lapatinib (LAP) is used since 2007 as dual TKI in the treatment of metastatic breast cancer and currently, it is used as an oral anticancer drug for the treatment of solid tumors such as breast and lung cancer. Although hepatotoxicity is its main side effect, it makes sense to investigate the ability of LAP to induce photosensitivity reactions bearing in mind that BRAF (serine/threonine-protein kinase B-Raf) inhibitors display a considerable phototoxic potential and that afloqualone, a quinazoline-marketed drug, causes photodermatosis. Metabolic bioactivation of LAP by CYP3A4 and CYP3A5 leads to chemically reactive N-dealkylated (N-LAP) and O-dealkylated (O-LAP) derivatives. In this context, the aim of the present work is to explore whether LAP and its N- and O-dealkylated metabolites can induce photosensitivity disorders by evaluating their photo(geno)toxicity through in vitro studies, including cell viability as well as photosensitized protein and DNA damage. As a matter of fact, our work has demonstrated that not only LAP, but also its metabolite N-LAP have a clear photosensitizing potential. They are both phototoxic and photogenotoxic to cells, as revealed by the 3T3 NRU assay and the comet assay, respectively. By contrast, the O-LAP does not display relevant photobiological properties. Remarkably, the parent drug LAP shows the highest activity in membrane phototoxicity and protein oxidation, whereas N-LAP is associated with the highest photogenotoxicity, through oxidation of purine bases, as revealed by detection of 8-Oxo-dG.

Vitamin A Deficiency and the Lung.

Vitamin A (all-trans-retinol) is a fat-soluble micronutrient which together with its natural derivatives and synthetic analogues constitutes the group of retinoids. They are involved in a wide range of physiological processes such as embryonic development, vision, immunity and cellular differentiation and proliferation. Retinoic acid (RA) is the main active form of vitamin A and multiple genes respond to RA signalling through transcriptional and non-transcriptional mechanisms. Vitamin A deficiency (VAD) is a remarkable public health problem. An adequate vitamin A intake is required in early lung development, alveolar formation, tissue maintenance and regeneration. In fact, chronic VAD has been associated with histopathological changes in the pulmonary epithelial lining that disrupt the normal lung physiology predisposing to severe tissue dysfunction and respiratory diseases. In addition, there are important alterations of the structure and composition of extracellular matrix with thickening of the alveolar basement membrane and ectopic deposition of collagen I. In this review, we show our recent findings on the modification of cell-junction proteins in VAD lungs, summarize up-to-date information related to the effects of chronic VAD in the impairment of lung physiology and pulmonary disease which represent a major global health problem and provide an overview of possible pathways involved.

Chronic alcohol exposure affects the cell components involved in membrane traffic in neuronal dendrites.

The specific traffic of the membrane components in neurons is a major requirement to establish and maintain neuronal domains-the axonal and the somatodendritic domains-and their polarized morphology. Unlike axons, dendrites contain membranous organelles, which are involved in the secretory pathway, including the endoplasmic reticulum, the Golgi apparatus and post-Golgi apparatus carriers, the cytoskeleton, and plasma membrane. A variety of molecules and factors are also involved in this process. Previous studies have shown that chronic alcohol exposure negatively affects several of these cell components, such as the Golgi apparatus or cytoskeleton in neurons. Yet very little information is available on the possible effects of this exposure on the remaining cell elements involved in intracellular trafficking in neurons, particularly in dendrites. By qualitative and quantitative electron microscopy, immunofluorescence and immunoblotting, we herein show that chronic exposure to moderate levels (30 mM) of ethanol in cultured neurons reduces the volume and surface density of the rough endoplasmic reticulum, and increases the levels of GRP78, a chaperone involved in endoplasmic reticulum stress. Ethanol also significantly diminishes the proportion of neurons that show an extension of Golgi into dendrites and dendritic Golgi outposts, a structure present exclusively in longer, thicker apical dendrites. Both Golgi apparatus types were also fragmented into a large number of cells. We also investigated the effect of alcohol on the levels of microtubule-based motor proteins KIF5, KIF17, KIFC2, dynein, and myosin IIb, responsible for transporting different cargoes in dendrites. Of these, alcohol differently affects several of them by lowering dynein and raising KIF5, KIFC2, and myosin IIb. These results, together with other previously published ones, suggest that practically all the protein trafficking steps in dendrites are altered to a greater or lesser extent by chronic alcohol exposure in neuronal cells, which may have negative repercussions for the development and maintenance of their polarized morphology and function.

Vitamin A deficiency disturbs collagen IV and laminin composition and decreases matrix metalloproteinase concentrations in rat lung. Partial reversibility by retinoic acid.

Vitamin A is essential for lung development and pulmonary cell differentiation. Its deficiency leads to altered lung structure and function and to basement membrane architecture and composition disturbances. Previously, we showed that lack of retinoids thickens the alveolar basement membrane and increases collagen IV, which are reversed by retinoic acid, the main biologically active vitamin A form. This study analyzed how vitamin A deficiency affects the subunit composition of collagen IV and laminin of lung basement membranes and pulmonary matrix metalloproteinase content, plus the recovering effect of all-trans-retinoic acid. Male weanling pups were fed a retinol-adequate/-deficient diet until 60 days old. A subgroup of vitamin-A-deficient pups received daily intraperitoneal all-trans-retinoic acid injections for 10 days. Collagen IV and laminin chain composition were modified in vitamin-A-deficient rats. The protein and mRNA contents of chains α1(IV), α3(IV) and α4(IV) increased; those of chains α2(IV) and α5(IV) remained unchanged; and the protein and mRNA contents of laminin chains α5, ß1 and γ1 decreased. The mRNA of laminin chains α2 and α4 also decreased. Matrix metalloproteinases 2 and 9 decreased, but the tissue inhibitors of metalloproteinases 1 and 2 did not change. Treating vitamin-A-deficient rats with retinoic acid reversed all alterations, but laminin chains α2, α4 and α5 and matrix metalloproteinase 2 remained low. In conclusion, vitamin A deficiency alters the subunit composition of collagen IV and laminin and the lung's proteolytic potential, which are partly reverted by retinoic acid. These alterations could contribute to impaired lung function and predispose to pulmonary disease.

Vitamin A deficiency increases protein catabolism and induces urea cycle enzymes in rats.

Chronic vitamin A deficiency induces a substantial delay in the rates of weight and height gain in both humans and experimental animals. This effect has been associated with an impaired nutrient metabolism and loss of body protein. Therefore, we analyzed the effect of vitamin A deficiency on endogenous proteolysis and nitrogen metabolism and its reversibility with all-trans retinoic acid (RA). Male weanling rats, housed in pairs, were pair-fed a vitamin A-deficient (VAD) or control diet until they were 60 d old. A group of deficient rats were further treated with daily intraperitoneal injections of all-trans RA for 10 d. Final body and tissue (i.e. liver and heart) weights were significantly lower and tissue:body weight ratios were similar in VAD rats and in controls. Conversely, the epididymal white fat:body weight ratio and the plasma concentrations of alanine aminotransferase and adiponectin were significantly higher in VAD rats, which also had hepatic macrovesicular lipid accumulations. Plasma and gastrocnemius muscle 3-methylhistidine, urine nitrogen, and plasma and urine urea concentrations were all significantly higher in the VAD group. The expression of the genes encoding urea cycle enzymes and their activities increased in VAD livers. These changes were partially reverted by all-trans RA. We propose that fuel partitioning in vitamin A deficiency may shift from fatty acids to protein catabolism as an energy source. Our results emphasize the importance of vitamin A on the energy balance control system and they provide an explanation for the role of vitamin A in protein turnover, development, and growth.

Vitamin A deficiency alters rat lung alveolar basement membrane: reversibility by retinoic acid.

Vitamin A is essential for lung development and pulmonary cell differentiation and its deficiency results in alterations of lung structure and function. Basement membranes (BMs) are also involved in those processes, and retinoic acid, the main biologically active form of vitamin A, influences the expression of extracellular matrix macromolecules. Therefore, we have analyzed the ultrastructure and collagen content of lung alveolar BM in growing rats deficient in vitamin A and the recovering effect of all-trans retinoic acid. Male weanling pups were fed a retinol-adequate or -deficient diet until they were 60 days old. A group of vitamin A-deficient pups were recovered by daily intraperitoneal injections of all-trans retinoic acid for 10 days. Alveolar BM in vitamin A-deficient rats doubled its thickness and contained irregularly scattered collagen fibrils. Immunocytochemistry revealed that these fibrils were composed of collagen I. Total content of both collagen I protein and its mRNA was greater in vitamin-deficient lungs. In agreement with the greater size of the BM the amount of collagen IV was also increased. Proinflammatory cytokines, IL-1alpha, IL-1beta and TNF-alpha, did not change, but myeloperoxidase and TGF-beta1 were increased. Treatment of vitamin A-deficient rats with retinoic acid reversed all the alterations, but the BM thickness recovered only partially. Retinoic acid recovering activity occurred in the presence of increasing oxidative stress. In conclusion, vitamin A deficiency results in alterations of the structure and composition of the alveolar BM which are probably mediated by TGF-beta1 and reverted by retinoic acid. These alterations could contribute to the impairment of lung function and predispose to pulmonary disease.

Vitamin A deficiency alters the structure and collagen IV composition of rat renal basement membranes.

Retinoids can modulate the expression of extracellular matrix (ECM) proteins with variable results depending on other contributing factors. Because changes in these proteins may alter the composition and impair the function of specialized ECM structures such as basement membranes (BMs), we studied the effects of vitamin A deficiency on renal BMs during the growing period. Newborn male rats were fed a vitamin A-deficient (VAD) diet for 50 d. The ultrastructure of renal BMs was analyzed by electron microscopy. Total collagen IV, the different alpha(IV) chains, matrix degrading metalloproteinases (MMP), and tissue inhibitors of metalloproteinases (TIMP) were quantified by immunocytochemistry and/or Western blotting. Tumor necrosis factor-alpha and interleukin-1beta were measured by ELISA. Semiquantitative RT-PCR was used for determining the steady-state levels for each alpha(IV) chain mRNA. VAD renal BMs showed an irregular thickening, particularly tubular BM. The total collagen IV content was increased, but there was a differential expression of the collagen IV chains. The protein amounts for alpha1(IV), alpha4(IV), and alpha5(IV) were similarly increased, whereas alpha2(IV) and alpha3(IV) were decreased. The levels of mRNA for each collagen IV chain changed in parallel with those of the corresponding protein. Both MMP2 and MMP9 were diminished, but no change was detected in TIMP1 or TIMP2. Our data indicate that nutritional VAD leads to alterations in the structure of renal BMs and to quantitative and qualitative variations in its collagen IV composition. These changes may be a factor predisposing to or resulting in kidney malfunction and renal disease.