1 Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio; and 2 Division of Gastroenterology, Departments of Physiology and Clinical Sciences, ...University of Liverpool, Liverpool, United Kingdom
Submitted 20 July 2006
; accepted in final form 20 October 2007
Cyclooxygenase-1 (Cox-1) contributes to gastric defense of healthy tissue, but the role in the protection of the gastric epithelium after minor, acute damage has been difficult to study in vivo. Using 710-nm two-photon light absorption to create microscopic gastric damage in anesthetized mice with the gastric mucosal surface surgically exposed and perfused on the microscope stage, the acute response of surface cells to injury could be monitored using in vivo microscopy within seconds after injury. Using exogenous (Cl-NERF) and endogenous fluorophores, extracellular pH and cell death were monitored in real time during the entire damage and repair cycle. Two-photon damage was initiated by scanning 200 µm 2 of gastric surface cells with high laser intensity, causing rapid bleaching of NAD(P)H fluorescence in optically targeted cells. In both Cox-1 +/– and Cox-1 –/– mice, a similar initial damage area expanded to include bystander epithelial cells over the next 2–5 min, with larger maximal damage noted in Cox-1 –/– mice. The maximal damage size seen in Cox-1 –/– mice could be reduced by exogenous dimethyl-PGE 2 . All damaged cells exfoliated, and the underlying epithelium was coincidently repaired over a time interval that was briefer in Cox-1 +/– (12 ± 2 min, n = 12) than in Cox-1 –/– (24 ± 4 min, n = 14) mice. Directly after damage, pH increased transiently in the juxtamucosal layer (maximal at 3–6 min). A smaller peak pH change was noted in Cox-1 –/– mice ( pH = 0.3 ± 0.04) than in Cox-1 +/– mice ( pH = 0.6 ± 0.2). Recovery to normal surface pH took longer in Cox-1 –/– mice (27 ± 5 min) than in Cox-1 +/– mice (12 ± 1 min). In conclusion, constitutive loss of Cox-1 leaves the gastric mucosa more prone to damage and slowed repair of microlesions.
autofluorescence; photodamage; laser scanning microscopy; two-photon microscopy; reduced nicotinamide adenine dinucleotide; surface pH; cyclooxygenase
Address for reprint requests and other correspondence: M. H. Montrose, Dept. of Molecular and Cellular Physiology, MSB 4207, Univ. of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267 (e-mail: mhm{at}uc.edu )
Although sterol carrier protein-2 (SCP-2) participates in the uptake and intracellular trafficking of cholesterol, its effect on “reverse cholesterol transport” has not been explored. As shown ...herein, SCP-2 expression inhibited high density lipoprotein (HDL)-mediated efflux of 3Hcholesterol and fluorescent 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3b-ol (NBD-cholesterol) up to 61 and 157%, respectively. Confocal microscopy of living cells allowed kinetic analysis of two intracellular pools of HDL-mediated NBD-cholesterol efflux: the highly fluorescent lipid droplet pool and the less fluorescent pool outside the lipid droplets, designated the cytoplasmic compartment. Both the whole cell and the cytoplasmic compartment exhibited two similar kinetic pools, the half-times of which were consistent with protein (tb12 near 1 min) and vesicular (td12 = 10–20 min) mediated sterol transfer. Although SCP-2 expression did not alter cytoplasmic sterol pool sizes, the rapid tb12 decreased 36%, while the slower td12 increased 113%. Lipid droplets also exhibited two kinetic pools of NBD-cholesterol efflux but with half-times over 200% shorter than those of the cytoplasmic compartment. The lipid droplet slower effluxing pool size and td12 were increased 48% and 115%, respectively, in SCP-2-expressing cells. Concomitantly, the level of the lipid droplet-specific adipose differentiation-related protein decreased 70%. Overall, HDL-mediated sterol efflux from L-cell fibroblasts reflected that of the cytoplasmic rather than lipid droplet compartment. SCP-2 differentially modulated sterol efflux from the two cytoplasmic pools. However, net efflux was determined primarily by inhibition of the slowly effluxing pool rather than by acceleration of the rapid protein-mediated pool. Finally, SCP-2 expression also inhibited sterol efflux from lipid droplets, an effect related to decreased adipose differentiation-related protein, a lipid droplet surface protein that binds cholesterol with high affinity.
Although the sterol carrier protein 2 (SCP-2) gene encodes for two proteins, almost nothing is known of the function and potential processing of the larger transcript corresponding to the 58 kDa ...sterol carrier protein-2/3-oxo-acyl-CoA thiolase (SCP-x), in intact cells. L-cell fibroblasts transfected with cDNA encoding for the 58 kDa SCP-x protein had a 4.5-fold increase in SCP-x mRNA transcript levels. Western blot analysis showed SCP-x protein expression reached 0.011% of total protein, representing a 4.1-fold increase over basal levels. Surprisingly, the 13.2 kDa SCP-2 protein also increased 2-fold in the transfected cells. This was consistent with part of the 58 kDa SCP-x being proteolytically processed to 13.2 kDa SCP-2 as there was no evidence of an mRNA transcript corresponding to a 13.2/15.2 kDa gene product in the transfected L-cell clones. Confocal immunofluorescence microscopy of transfected L-cells showed that SCP-x/SCP-2 co-localized in highest concentration with catalase in peroxisomes, but significant amounts appeared extra-peroxisomal. Overexpression of SCP-x significantly altered cholesterol uptake and metabolism. Uptake of exogenous 3Hcholesterol and total cholesterol mass were increased 1.9- and 1.4-fold, respectively, in SCP-x expressors. Although cholesterol ester mass was unaltered, incorporation of exogenous 3Hcholesterol and 3Holeic acid into cholesteryl esters increased 2.3- and 2.5-fold, respectively. These results from intact cells suggest the 13.2 kDa SCP-2 can arise from the larger SCP-2 gene product and indicate a role for the 58 kDa SCP-x protein in cholesterol uptake and intracellular cycling.—Atshaves, B. P., A. D. Petrescu, O. Starodub, J. B. Roths, A. B. Kier, and F. Schroeder. Expression and intracellular processing of the 58 kDa sterol carrier protein-2/3-oxoacyl-CoA thiolase in transfected mouse L-cell fibroblasts. J. Lipid Res. 1999. 40: 610–622.
Although unesterified long chain fatty acids interact with peroxisome proliferator-activated receptors to initiate transcription within the nucleus, almost nothing is known regarding factors ...regulating long chain fatty acid distribution to the nucleus of living cells. The possibility that the liver fatty acid-binding protein (L-FABP) may function in this role was addressed in transfected L-cell fibroblasts overexpressing L-FABP using a series of fluorescent fatty acids differing in chain length and unsaturation. After 30 min of incubation, oxidation of BODIPY-, NBD-, and cis-parinaric acids was undetectable in L-cells. Likewise, L-cells very poorly esterified these fluorescent fatty acids in the following order: 0% BODIPY-C5, NBD-C6 (short chain length) < 0-3% NBD-C18, BODIPY-C16, cis-parinaric acid (long chain length) < 11% BODIPY-C12 (medium chain length). Real time confocal and multiphoton laser scanning microscopy (CLSM and MPLSM) showed that these fluorescent fatty acids were generally taken up in the following order: long chain (BODIPY-C16, NBD-C18) > medium chain (BODIPY-C12) short chain (BODIPY-C5, NBD-C6). The fluorescent fatty acids were imaged in the nucleus, primarily associated with the nuclear envelope, at levels about 2-3-fold lower than outside the nucleus. CLSM and MPLSM showed that L-FABP expression enhanced by 2-4-fold the initial rate and/or average maximal uptake of the long and medium chain but not the short chain fluorescent fatty acids in living cells. Furthermore, L-FABP expression increased the targeting of long and medium but not short chain fluorescent fatty acids to the nucleus by 2.9-4.4-fold and increased the proportion (i.e. nuclear:cytoplasm ratio) of medium and long chain but not short chain fatty acids by 2-3.6-fold. In summary, these results showed for the first time the presence of unesterified fatty acids in the nucleus of living cells and demonstrated that expression of a fatty acid-binding protein, L-FABP, specifically enhanced uptake and intracellular targeting of long and medium chain fatty acids to the nucleus.
Although unesterified long chain fatty acids interact with peroxisome proliferator-activated receptors to initiate transcription
within the nucleus, almost nothing is known regarding factors ...regulating long chain fatty acid distribution to the nucleus
of living cells. The possibility that the liver fatty acid-binding protein (L-FABP) may function in this role was addressed
in transfected L-cell fibroblasts overexpressing L-FABP using a series of fluorescent fatty acids differing in chain length
and unsaturation. After 30 min of incubation, oxidation of BODIPY-, NBD-, and cis- parinaric acids was undetectable in L-cells. Likewise, L-cells very poorly esterified these fluorescent fatty acids in the
following order: 0% BODIPY-C5, NBD-C6 (short chain length) < 0â3% NBD-C18, BODIPY-C16, cis- parinaric acid (long chain length) < 11% BODIPY-C12 (medium chain length). Real time confocal and multiphoton laser scanning
microscopy (CLSM and MPLSM) showed that these fluorescent fatty acids were generally taken up in the following order: long
chain (BODIPY-C16, NBD-C18) > medium chain (BODIPY-C12) â« short chain (BODIPY-C5, NBD-C6). The fluorescent fatty acids were
imaged in the nucleus, primarily associated with the nuclear envelope, at levels about 2â3-fold lower than outside the nucleus.
CLSM and MPLSM showed that L-FABP expression enhanced by 2â4-fold the initial rate and/or average maximal uptake of the long
and medium chain but not the short chain fluorescent fatty acids in living cells. Furthermore, L-FABP expression increased
the targeting of long and medium but not short chain fluorescent fatty acids to the nucleus by 2.9â4.4-fold and increased
the proportion ( i.e. nuclear:cytoplasm ratio) of medium and long chain but not short chain fatty acids by 2â3.6-fold. In summary, these results
showed for the first time the presence of unesterified fatty acids in the nucleus of living cells and demonstrated that expression
of a fatty acid-binding protein, L-FABP, specifically enhanced uptake and intracellular targeting of long and medium chain
fatty acids to the nucleus.
Although it is hypothesized that long-chain fatty acyl CoAs (LCFA-CoAs) and long-chain fatty acids (LCFAs) regulate transcription in the nucleus, little is known regarding factors that determine the ...distribution of these ligands to nuclei of living cells. Immunofluorescence colocalization showed that liver fatty acid-binding protein (L-FABP; binds LCFA-CoA as well as LCFA) significantly colocalized with PPARα in nuclei of transfected L-cell fibroblasts. Colocalization with a DNA binding dye (SYTO59) revealed that, within the nucleus of control L-cells, the nonhydrolyzable fluorescent LCFA-CoA (BODIPY-C16-S-S-CoA) was distributed primarily in a punctate pattern throughout the nucleoplasm, while nonmetabolizable fluorescent LCFAs (BODIPY-C16 and BODIPY-C12) were localized primarily near the nuclear envelope membranes. L-FABP overexpression selectively increased the targeting of BODIPY-C16-S-S-CoA by 1.9- and 2.7-fold into the nuclear membrane and nucleoplasm, respectively. L-FABP also increased the targeting of fluorescent LCFAs (especially long-chain-length BODIPY-C16) by 1.7-fold to the nuclear membrane and 7.4-fold into the nucleoplasm. A cis-parinaric acid displacement assay showed that L-FABP bound BODIPY-C12 and BODIPY-C16 with K is of 10.1 ± 2.5 and 20.7 ± 1.5 nM, respectively, in the same range as naturally occurring LCFAs. Finally, solid-phase extraction and HPLC analysis revealed that, depending on the fatty acid content of the culture medium, L-FABP expression also increased the cellular LCFA-CoA pool size and altered the LCFA-CoA acyl chain composition. Thus, L-FABP may function as a carrier for selectively enhancing the distribution of LCFA-CoA, as well as LCFA, to nuclei for potential interaction with nuclear receptors.
Since its discovery three decades ago, sterol carrier protein-2 (SCP-2) has remained a fascinating protein whose physiological function in lipid metabolism remains an enigma. Its multiple proposed ...functions arise from its complex gene structure, post-translational processing, intracellular localization, and ligand specificity. The SCP-2 gene has two initiation sites coding for proteins that share a common 13 kDa SCP-2 C-terminus: (1) One site codes for 58 kDa SCP-x which is partially post-translationally cleaved to 13 kDa SCP-2 and a 45 kDa protein. (2) A second site codes for 15 kDa pro-SCP-2 which is completely post-translationally cleaved to 13 kDa SCP-2. Very little is yet known regarding how the relative proportions of the two transcripts are regulated. Although all three proteins contain a C-terminal SKL peroxisomal targeting sequence, it is unclear why all three proteins are not exclusively localized in peroxisomes. However, the recent demonstration that the SCP-2 N-terminal presequence in pro-SCP-2 dramatically modulated the intracellular targeting coded by the C-terminal peroxisomal targeting sequence may account for the observation that as much as half of total SCP-2 is localized outside the peroxisome. The tertiary and secondary structure of the 13 kDa SCP-2, but not that of 15 kDa pro-SCP-2 and 58 kDa SCP-x, are now resolved. Increasing evidence suggests that the 58 kDa SCP-x and 45 kDa proteins are peroxisomal 3-ketoacyl-CoA-thiolases involved in the oxidation of branched chain fatty acids. Since 15 kDa pro-SCP-2 is post-translationally completely cleaved to 13 kDa SCP-2, relatively little attention has been focused on this protein. Finally, although the 13 kDa SCP-2 is the most studied of these proteins, because it exhibits diversity of its ligand partners (fatty acids, fatty acyl CoAs, cholesterol, phospholipids), new potential physiological function(s) are still being proposed and questions regarding potential compensation by other proteins with overlapping specificity are only beginning to be resolved.
Cellular cholesterol homeostasis is a balance of influx, catabolism and synthesis, and efflux. Unlike vascular lipoprotein cholesterol transport, intracellular cholesterol trafficking is only ...beginning to be resolved. Exogenous cholesterol and cholesterol ester enter cells via the low-density lipoprotein (LDL) receptor/lysosomal and less so by nonvesicular, high-density lipoprotein (HDL) receptor/caveolar pathways. However, the mechanism(s) whereby cholesterol enters the lysosomal membrane, translocates, and transfers out of the lysosome to the cell interior are unknown. Likewise, the steps whereby cholesterol enters the cytofacial leaflet of the plasma membrane caveolae, rapidly translocates, leaves the exofacial leaflet, and transfers to extracellular HDL are unclear. Increasing evidence obtained with model and isolated cell membranes, transfected cells, genetic mutants, and gene-ablated mice suggests that proteins such as caveolin, sterol carrier protein-2 (SCP-2), Niemann-Pick C1 protein, steroidogenic acute regulatory protein (StAR), and other intracellular proteins mediate intracellular cholesterol transfer. While these proteins bind cholesterol and/or interact with cholesterol-rich membrane microdomains (e.g., caveolae, rafts, and annuli), their relative contributions to direct molecular versus vesicular cholesterol transfer remain to be resolved. The formation, regulation, and role of membrane microdomains in regulating cholesterol uptake/efflux and trafficking are unclear. Some cholesterol-binding proteins exert opposing effects on cellular cholesterol uptake/efflux, transfer of cholesterol out of the lysosomal membrane, and/or intracellular cholesterol trafficking to select membranous organelles. Resolving these cholesterol pathways and the role of membrane cholesterol microdomains is essential to our understanding not only of processes that affect cholesterol metabolism, but also of the abnormal regulation that may lead to disease (diabetes, obesity, atherosclerosis, neutral lipid storage, Niemann-Pick C, congenital lipoid adrenal hyperplasia, etc.).
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