Tissue-specific sorting of the human LDL receptor in polarized epithelia of transgenic mice.

The distribution of human low density lipoprotein (LDL) receptors was studied by immunofluorescence and immunoelectron microscopy in epithelial cells of transgenic mice that express high levels of receptors under control of the metallothionein-I promoter. In hepatocytes and intestinal epithelial cells, the receptors were confined to the basal and basolateral surfaces, respectively. Very few LDL receptors were present in coated pits or intracellular vesicles. In striking contrast, in the epithelium of the renal tubule the receptors were present on the apical (lumenal) surface where they appeared to be concentrated at the base of microvilli and were abundant in vesicles of the endocytic recycling pathway. Intravenously administered LDL colloidal gold conjugates bound to the receptors on hepatocyte microvilli and were slowly internalized, apparently through slow migration into coated pits. We conclude that (a) sorting of LDL receptors to the surface of different epithelial cells varies with each tissue; and (b) in addition to a signal for clustering in coated pits, the LDL receptor may contain a signal for retention in noncoated membrane that is manifest in hepatocytes and intestinal epithelial cells, but not in renal epithelial cells or cultured human fibroblasts.

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Location and regulation of low-density lipoprotein receptors in intestinal epithelium

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1995.269.1.g60 ◽

1995 ◽

Vol 269 (1) ◽

pp. G60-G72 ◽

Cited By ~ 2

Author(s):

L. G. Fong ◽

S. E. Fujishima ◽

M. C. Komaromy ◽

Y. K. Pak ◽

J. L. Ellsworth ◽

...

Keyword(s):

Epithelial Cells ◽

Ethinyl Estradiol ◽

Intestinal Epithelial Cells ◽

Low Density Lipoprotein ◽

Ldl Receptor ◽

Density Lipoprotein ◽

Low Density ◽

Intestinal Epithelial ◽

Ldl Receptors ◽

Receptor Mrna

The expression, distribution, and some aspects of the regulation of low-density lipoprotein (LDL) receptors in rat intestinal epithelial cells were examined. Cells prepared by a perfusion technique provided a pure preparation of epithelial cells and could be manipulated to produce crypt-villus units or villi alone. On a total protein basis, the abundance of LDL receptors in villus cell membranes was half that in hepatic membranes. The level of receptors in both tissues was reduced by feeding an atherogenic diet but was increased only in the liver by ethinyl estradiol-induced hypocholesterolemia. The level of LDL receptor mRNA in intestinal epithelial cells was somewhat lower than in liver. Regulation of LDL receptor mRNA was similar to that of protein. Judged by the ratio of mRNA in villus cells to the villus-crypt unit and nuclear run-on assay for LDL receptor gene transcription, we conclude that LDL receptor mRNA is produced in the villus cells. The effect of fat feeding was regulated at the level of transcription. Expression in villus cells in ileum was severalfold higher than in jejunum and higher than in the liver. Together the results suggest serum cholesterol level is not the prime determinant of LDL receptor level in intestine, but LDL degradation in this organ may be regulated by factors in the lumen.

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Modulation of regulatory oxysterol formation and low density lipoprotein suppression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity by ketoconazole. A role for cytochrome P-450 in the regulation of HMG-CoA reductase in rat intestinal epithelial cells.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)83919-4 ◽

1986 ◽

Vol 261 (18) ◽

pp. 8348-8356 ◽

Cited By ~ 16

Author(s):

A Gupta ◽

R C Sexton ◽

H Rudney

Keyword(s):

Epithelial Cells ◽

Intestinal Epithelial Cells ◽

Reductase Activity ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Low Density ◽

Intestinal Epithelial ◽

Hmg Coa Reductase ◽

Coa Reductase ◽

Cytochrome P 450

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Inefficient internalization of receptor-bound low density lipoprotein in human carcinoma A-431 cells.

The Journal of Cell Biology ◽

10.1083/jcb.88.2.441 ◽

1981 ◽

Vol 88 (2) ◽

pp. 441-452 ◽

Cited By ~ 57

Author(s):

R G Anderson ◽

M S Brown ◽

J L Goldstein

Keyword(s):

Cho Cells ◽

High Efficiency ◽

Low Density Lipoprotein ◽

Human Fibroblasts ◽

Density Lipoprotein ◽

Cell Types ◽

Low Density ◽

Coated Pits ◽

Human Carcinoma ◽

Ldl Receptors

Human epithelioid carcinoma A-431 cells are known to express unusually large numbers of receptors for the polypeptide hormone epidermal growth factor. The current studies demonstrate that this cell line also expresses 5- to 10-fold more low density lipoprotein (LDL) receptors per cell than either human fibroblasts or Chinese hamster ovary (CHO) cells. As visualized with an LDL-ferritin conjugate, the LDL receptors in A-431 cells appeared in clusters that were distributed uniformly over the cell surface, occurring over flat regions of the membrane as well as over the abundant surface extensions. Only 4% of the LDL receptors were located in coated pits. The LDL receptors in A-431 cells showed the same affinity and specificity as the LDL receptors in human fibroblasts and other cell types. In addition, they were subject to feedback regulation by sterols in the same manner as the LDL receptors in other cells. However, in contrast to other cell types in which the receptor-bound LDL is internalized with high efficiency, in the A-431 cells only a small fraction of the receptor-bound LDL entered the cell. In CHO cells approximately 66% of the LDL receptors were located over coated regions of membrane, and the efficiency of LDL internalization was correspondingly 10-fold higher than in A-431 cells. These findings support the concept that the rate of LDL internalization is proportional to the number of LDL receptors in coated pits and that the inefficiency of internalization in the A-431 cells is caused by a limitation in the ability of these cells to incorporate their LDL receptors into coated pits.

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A mutation and an antibody that affect chemical cross-linking of low-density lipoprotein receptors on human fibroblasts

Biochemical Journal ◽

10.1042/bj2890569 ◽

1993 ◽

Vol 289 (2) ◽

pp. 569-573 ◽

Cited By ~ 5

Author(s):

D D Patel ◽

A K Soutar ◽

B L Knight

Keyword(s):

Tertiary Structure ◽

Low Density Lipoprotein ◽

Human Fibroblasts ◽

Surface Membrane ◽

Ldl Receptor ◽

Density Lipoprotein ◽

Cross Linking ◽

Low Density ◽

Ldl Receptors ◽

Surface Receptors

Treatment of normal fibroblasts with the bifunctional cross-linking reagent DTSSP [3,3′-dithiobis(sulphosuccinimidylpropionate)] at 4 degrees C converted approximately 40% of the cell-surface low-density lipoprotein (LDL) receptors into a high-M(r) form, thought to represent receptor dimers. Preincubation of the cells with anti-(LDL receptor) monoclonal antibody 10A2 increased the proportion of surface receptors in the high-M(r) form after treatment with DTSSP at 4 degrees C to over 70%. Preincubation with LDL did not affect the proportion cross-linked, but prevented the increase produced by antibody 10A2. Cross-linking at 37 degrees C was less efficient than at 4 degrees C and was not affected by preincubation with antibody 10A2. Surface LDL receptors on fibroblasts from the homozygous familial hypercholesterolaemic subject MM were not cross-linked by DTSSP, confirming that the mutation had produced a change in the conformation of the receptor molecule. Taken together, the results suggest that normal LDL receptors on at least one region of the surface membrane may be loosely associated in some form of multimeric array which alters its alignment differently in response to antibody 10A2 and to cooling. Mutations that alter the tertiary structure of the receptors could affect LDL binding by disturbing the arrangement of the array.

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Potassium-dependent assembly of coated pits: new coated pits form as planar clathrin lattices.

The Journal of Cell Biology ◽

10.1083/jcb.103.6.2619 ◽

1986 ◽

Vol 103 (6) ◽

pp. 2619-2627 ◽

Cited By ~ 95

Author(s):

J M Larkin ◽

W C Donzell ◽

R G Anderson

Keyword(s):

Critical Size ◽

Shape Change ◽

Low Density Lipoprotein ◽

Human Fibroblasts ◽

Density Lipoprotein ◽

Coated Pits ◽

Ldl Receptors ◽

Membrane Surfaces ◽

Membrane Shape ◽

K Depletion

Previous studies have shown that when human fibroblasts are depleted of intracellular K+, coated pits disappear from the cell surface and the receptor-mediated endocytosis of low density lipoprotein (LDL) is inhibited. We have now used the K+ depletion protocol to study several aspects of coated pit function. First, since coated pits rapidly form when K+-depleted fibroblasts are incubated in the presence of 10 mM KCl, we studied the sequence of assembly of coated pits as visualized in carbon-platinum replicas of inner membrane surfaces from cells that had been incubated in the presence of K+ for various times. New coated pits initially appeared as planar clathrin lattices that increased in size by the formation of polygons at the margin of the lattice. Once the lattice reached a critical size it invaginated to form coated vesicles. Second, we determined that LDL-ferritin can induce clustering of LDL receptors over noncoated membrane on the surface of K+-depleted fibroblasts; however, when these cells are subsequently incubated in the presence of K+, these clusters become associated with newly formed coated pits and are internalized. Finally, we determined that K+ depletion inhibits the assembly of coated pits, but that existing coated pits in K+-depleted cells are able to internalize LDL. These results suggest that the clathrin lattice of coated pits is actively involved in membrane shape change during endocytosis and that the structural proteins of the lattice are cyclically assembled and disassembled in the process.

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Post-transcriptional regulation of Wnt co-receptor LRP6 and RNA-binding protein HuR by miR-29b in intestinal epithelial cells

Biochemical Journal ◽

10.1042/bcj20160057 ◽

2016 ◽

Vol 473 (11) ◽

pp. 1641-1649 ◽

Cited By ~ 9

Author(s):

Yanwu Li ◽

Gang Chen ◽

Jun-Yao Wang ◽

Tongtong Zou ◽

Lan Liu ◽

...

Keyword(s):

Epithelial Cells ◽

Rna Binding ◽

Intestinal Epithelial Cells ◽

Binding Protein ◽

Low Density Lipoprotein ◽

Rna Binding Protein ◽

Density Lipoprotein ◽

Transcriptional Level ◽

Intestinal Epithelial ◽

Repressive Effect

MicroRNAs (miRNAs) control gene expression by binding to their target mRNAs for degradation and/or translation repression and are implicated in many aspects of cellular physiology. Our previous study shows that miR-29b acts as a biological repressor of intestinal mucosal growth, but its exact downstream targets remain largely unknown. In the present study, we found that mRNAs, encoding Wnt co-receptor LRP6 (low-density lipoprotein-receptor-related protein 6) and RNA-binding protein (RBP) HuR, are novel targets of miR-29b in intestinal epithelial cells (IECs) and that expression of LRP6 and HuR is tightly regulated by miR-29b at the post-transcriptional level. miR-29b interacted with both Lrp6 and HuR mRNAs via their 3′-UTRs and inhibited LRP6 and HuR expression by destabilizing Lrp6 and HuR mRNAs and repressing their translation. Studies using heterologous reporter constructs revealed a greater repressive effect of miR-29b through a single binding site in the Lrp6 or HuR 3′-UTR, whereas deletion mutation of this site prevented miR-29b-induced repression of LRP6 and HuR expression. Repression of HuR by miR-29b in turn also contributed to miR-29b-induced LRP6 inhibition, since ectopic overexpression of HuR in cells overexpressing miR-29b restored LRP6 expression to near normal levels. Taken together, our results suggest that miR-29b inhibits expression of LRP6 and HuR post-transcriptionally, thus playing a role in the regulation of IEC proliferation and intestinal epithelial homoeostasis.

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Positive cross talk between protein kinase D and β-catenin in intestinal epithelial cells: impact on β-catenin nuclear localization and phosphorylation at Ser552

AJP Cell Physiology ◽

10.1152/ajpcell.00302.2015 ◽

2016 ◽

Vol 310 (7) ◽

pp. C542-C557 ◽

Cited By ~ 9

Author(s):

Jia Wang ◽

Liang Han ◽

James Sinnett-Smith ◽

Li-Li Han ◽

Jan V. Stevens ◽

...

Keyword(s):

Protein Kinase ◽

Epithelial Cells ◽

Transgenic Mice ◽

Nuclear Localization ◽

Cross Talk ◽

Intestinal Epithelial Cells ◽

Dependent Manner ◽

Intestinal Epithelial ◽

Ang Ii ◽

Potent Inducer

Given the fundamental role of β-catenin signaling in intestinal epithelial cell proliferation and the growth-promoting function of protein kinase D1 (PKD1) in these cells, we hypothesized that PKDs mediate cross talk with β-catenin signaling. The results presented here provide several lines of evidence supporting this hypothesis. We found that stimulation of intestinal epithelial IEC-18 cells with the G protein-coupled receptor (GPCR) agonist angiotensin II (ANG II), a potent inducer of PKD activation, promoted endogenous β-catenin nuclear localization in a time-dependent manner. A significant increase was evident within 1 h of ANG II stimulation ( P < 0.01), peaked at 4 h ( P < 0.001), and declined afterwards. GPCR stimulation also induced a marked increase in β-catenin-regulated genes and phosphorylation at Ser552 in intestinal epithelial cells. Exposure to preferential inhibitors of the PKD family (CRT006610 or kb NB 142-70) or knockdown of the isoforms of the PKD family prevented the increase in β-catenin nuclear localization and phosphorylation at Ser552 in response to ANG II. GPCR stimulation also induced the formation of a complex between PKD1 and β-catenin, as shown by coimmunoprecipitation that depended on PKD1 catalytic activation, as it was abrogated by cell treatment with PKD family inhibitors. Using transgenic mice that express elevated PKD1 protein in the intestinal epithelium, we detected a marked increase in the localization of β-catenin in the nucleus of crypt epithelial cells in the ileum of PKD1 transgenic mice, compared with nontransgenic littermates. Collectively, our results identify a novel cross talk between PKD and β-catenin in intestinal epithelial cells, both in vitro and in vivo.

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Effect of cell density on binding and uptake of low density lipoprotein by human fibroblasts.

The Journal of Cell Biology ◽

10.1083/jcb.83.3.588 ◽

1979 ◽

Vol 83 (3) ◽

pp. 588-594 ◽

Cited By ~ 54

Author(s):

H S Kruth ◽

J Avigan ◽

W Gamble ◽

M Vaughan

Keyword(s):

Cell Density ◽

Low Density Lipoprotein ◽

Human Fibroblasts ◽

Density Lipoprotein ◽

Low Density ◽

Cellular Lipid ◽

Cholesterol Accumulation ◽

Ldl Receptors ◽

Ldl Binding ◽

In Cells

The effect of cell density on low density lipoprotein (LDL) binding by cultured human skin fibroblasts was investigated. Bound LDL was visualized by indirect immunofluorescence. Cellular lipid and cholesterol were monitored by fluorescence in cells stained with phosphine 3R and filipin, respectively. LDL binding and lipid accumulation were compared in cells in stationary and exponentially growing cultures, in sparsely and densely plated cultures, in wounded and non-wounded areas of stationary cultures, and in stationary cultures with and without the addition of lipoprotein-deficient serum. We conclude that LDL binding and cholesterol accumulation induced by LDL are influenced by cell density. It appears that, compared to rapidly growing cells, quiescent (noncycling) human fibroblasts exhibit fewer functional LDL receptors.

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Characterization of a family of gamma-ray-induced CHO mutants demonstrates that the ldlA locus is diploid and encodes the low-density lipoprotein receptor

Molecular and Cellular Biology ◽

10.1128/mcb.6.9.3268-3277.1986 ◽

1986 ◽

Vol 6 (9) ◽

pp. 3268-3277

Author(s):

R D Sege ◽

K F Kozarsky ◽

M Krieger

Keyword(s):

Gamma Irradiation ◽

Cho Cells ◽

Gamma Ray ◽

Receptor Gene ◽

Low Density Lipoprotein ◽

Chinese Hamster ◽

Ldl Receptor ◽

Density Lipoprotein ◽

Low Density ◽

Ldl Receptors

The ldlA locus is one of four Chinese hamster ovary (CHO) cell loci which are known to be required for the synthesis of functional low-density lipoprotein (LDL) receptors. Previous studies have suggested that the ldlA locus is diploid and encodes the LDL receptor. To confirm this assignment, we have isolated a partial genomic clone of the Chinese hamster LDL receptor gene and used this and other nucleic acid and antibody probes to study a family of ldlA mutants isolated after gamma-irradiation. Our analysis suggests that there are two LDL receptor alleles in wild-type CHO cells. Each of the three mutants isolated after gamma-irradiation had detectable deletions affecting one of the two LDL receptor alleles. One of the mutants also had a disruption of the remaining allele, resulting in the synthesis of an abnormal receptor precursor which was not subject to Golgi-associated posttranslational glycoprotein processing. The correlation of changes in the expression, structure, and function of LDL receptors with deletions in the LDL receptor genes in these mutants directly demonstrated that the ldlA locus in CHO cells is diploid and encodes the LDL receptor. In addition, our analysis suggests that CHO cells in culture may contain a partial LDL receptor pseudogene.

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