Nickel is a specific antagonist for the catabolism of activated alpha 2-macroglobulin.

The multifunctional low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) binds and degrades several ligands involved in protease and lipoprotein metabolism. We previously reported that nickel (Ni2+) specifically inhibits the binding of activated alpha 2-macroglobulin (alpha 2 M*) at 4 degrees C to LRP and had no effect on the binding of other ligands to the receptor (Hussain et al. (1995) Biochem. 34, 16074-16081). In the current investigation, we have examined the effect of Ni2+ on the catabolism of 125 I-labeled alpha 2M*, receptor-associated protein (RAP) and lactoferrin at physiologic temperatures by fibroblasts. Nickel completely inhibited the degradation of alpha 2M* over a wide range of concentrations (0.3-2.4 nM); 50% inhibition for the degradation of 1.2 nM alpha 2M* was observed at 0.5 mM Ni2+. Furthermore, nickel inhibited the binding, internalization and degradation of 125I-alpha 2M* in a dose- and time-dependent manner. In contrast, the degradation of several concentrations of 125I-RAP by fibroblasts was not affected by different amounts of Ni2+ for various times. Similarly, Ni2+ did not inhibit the degradation of lactoferrin either before or after treating the cells with heparitinase to remove cell-surface proteoglycans. The degradation of lactoferrin was, however, inhibited by the RAP indicating that lactoferrin degradation was mediated by the LRP. These data suggest that Ni2+ is a specific inhibitor for the degradation of alpha 2M*.

Up-regulation of the low density lipoprotein receptor-related protein by dexamethasone in HepG2 cells.

Dexamethasone has been shown to decrease the expression of the low density lipoprotein (LDL) receptor, but its effect on other members of the LDL receptor family is not known. We studied the effect of dexamethasone in HepG2 cells on the expression of the LDL receptor family members using radiolabeled receptor associated protein (RAP) which binds to all the members of the family. Treatment of HepG2 cells with increasing concentrations of dexamethasone resulted in a 2-fold increase in the binding and degradation of RAP. To identify the receptor responsible for the increased binding and degradation of RAP, we used specific ligands. For LDL receptor, we used LDL itself. For the LDL receptor-related protein/alpha 2-macroglobulin receptor, we used activated alpha 2-macroglobulin. The binding of LDL to HepG2 cells was decreased, whereas binding and degradation of activated alpha 2-macroglobulin was increased by 2-fold suggesting that dexamethasone increased LRP expression. Increased LRP expression was positively correlated with the increase in the steady-state levels and transcript numbers of the LRP mRNA; no changes in RAP or gamma-actin mRNA levels were observed. Increased mRNA levels were not due to an increased rate of transcription of the gene as assessed by nuclear run-on experiments. These studies indicate that dexamethasone increases cell-surface LRP activity in HepG2 cells by increasing the steady state mRNA levels and suggest that post-transcriptional mechanisms play a role in controlling LRP mRNA levels.

Chylomicron assembly and catabolism: role of apolipoproteins and receptors.

Chylomicrons are lipoproteins synthesized exclusively by the intestine to transport dietary fat and fat-soluble vitamins. Synthesis of apoB48, a translational product of the apob gene, is required for the assembly of chylomicrons. The apob gene transcription in the intestine results in 14 and 7 kb mRNAs. These mRNAs are post-transcriptionally edited creating a stop codon. The edited mRNAs chylomicrons from the shorter apoB48 peptide remains to be elucidated. In addition, the roles of proteins involved in the assembly pathway, e.g. apobec-1, MTP and apoA-IV, needs to be studied. Cloning of enzymes involved in the intestinal biosynthesis of triglycerides will be crucial to fully appreciate the assembly of chylomicrons. There is a need for cell culture and transgenic animal models that can be used for intestinal lipoprotein assembly. The catabolism of chylomicrons is far more complex and efficient than the catabolism of VLDL. Even though the major steps involved in the catabolism of chylomicrons are now known, the determinants for apolipoprotein exchange, processing of remnants in the space of Disse, as well as the mechanism of uptake of these particles by extra-hepatic tissue needs further exploration.

Nickel is a specific inhibitor for the binding of activated alpha 2-macroglobulin to the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.

The low density receptor-related protein/alpha 2-macroglobulin receptor (LRP/alpha 2-MR) binds to several ligands involved in lipoprotein and protease clearance. The receptor-associated protein (RAP) inhibits the binding of all known ligands. We studied the inhibition by Ni2+ of the binding of different ligands to cells and to the purified LRP/alpha 2-MR. Ni2+ inhibited all of the specific binding of radiolabeled methylamine-activated alpha 2-macroglobulin (125I-alpha 2-M*) to rabbit aortic smooth muscle cells (SMC), rat hepatoma Fu5AH, and mouse fibroblast L cells. Ni2+ also inhibited the binding of trypsin-activated alpha 2-macroglobulin to SMC but did not affect the binding of RAP, Pseudomonas exotoxin A, or low-density lipoproteins. The inhibition of alpha 2-M* binding by Ni2+ was not due to its interaction with alpha 2-M*. Preincubation of SMC with Ni2+ followed by ligand binding suggested that Ni2+ binds to cell-surface molecules and inhibits the binding of alpha 2-M* but does not affect RAP binding. Most of the binding of alpha 2-M* to SMC was due to its binding to the LRP/alpha 2-MR, as opposed to the recently described signaling receptor, as demonstrated by the inhibition of this binding by the RAP. Moreover, the inhibition of alpha 2-M* binding to the LRP/alpha 2-MR by Ni2+ was demonstrated using purified receptor immobilized on microtiter plates. Two to three molecules of 63Ni2+ bound to the immobilized receptor with equal affinity but not to alpha 2-M*. The specific binding of alpha 2-M* to the immobilized receptor was inhibited in the presence of nickel.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of recombinant human apoB-48-containing lipoproteins in rat hepatoma McA-RH7777 cells transfected with apoB-48 cDNA. Overexpression of apoB-48 decreases synthesis of endogenous apoB-100.

We studied the effect of overexpression of apolipoprotein (apo) B-48 on the synthesis and secretion of endogenous apoB-100 in rat hepatoma McA-RH7777 cell lines stably transfected with human apoB-48 cDNA under the control of the cytomegalovirus promoter. Three cell lines that secrete 40 to 60 ng human apoB.mg cell protein-1.h-1 were used. The recombinant human apoB-48 exhibited physicochemical characteristics (buoyant density, 1.06 to 1.21 g/mL; beta-electrophoretic mobility and diameters, 16 to 20 nm) indistinguishable from those of endogenous rat apoB-48. Overexpression of the recombinant human apoB-48 resulted in a 50% decrease in the secretion of endogenous apoB-100 but did not affect the secretion of apoE or apoA-I. Several possible mechanisms for the decreased secretion of apoB-100 were evaluated. First, recruitment of lipids into lipoproteins was shown to be unaffected since no major changes in the physicochemical properties of apoB-100-containing lipoproteins were observed. Second, the intracellular degradation of apoB-100 was not altered as the intracellular retention half-time and secretion efficiency remained unaffected by apoB-48 overexpression. Third, the posttranslational regulatory mechanisms for apoB-100 remained normal, as demonstrated by a twofold increase in apoB-100 secretion after supplementation with oleic acid. Unexpectedly, a 35% to 50% decrease in the steady-state synthesis of endogenous apoB-100 was observed in apoB-48-transfected cells compared with control cells. These data suggested that decreased secretion of apoB-100 was secondary to decreased synthesis. The decreased apoB-100 synthesis was not due to decreased steady-state levels of rat apoB-100 mRNA. These results suggest that overexpression of recombinant human apoB-48 may interfere with posttranscriptional events, possibly at the translation-translocation level, and decrease translational yield of apoB-100. These posttranscriptional events prior to the complete synthesis of the apoB-100 polypeptide can be important in the control of apoB-100 secretion.

Decreased expression of the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor in invasive cell clones derived from human prostate and breast tumor cells.

The importance of receptors involved in the clearance of proteases and protease/inhibitor complexes in tumor invasion is unknown. We studied the expression of the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP/alpha 2-MR), the major receptor involved in the clearance of protease/inhibitor complexes, in invasive and noninvasive subclones derived from tumor cells. The receptor activity was 2- to 3-fold lower in invasive subclones compared to noninvasive subclones derived from human prostate PC-3 and DU 145 and melanoma A2058 cells. The receptor activity was decreased in breast cancer (MCF-10A) cells transfected with mutated Ha-ras compared to nontransfected cells. Furthermore, invasive cells derived from ras-transfected MCF-10A cells expressed lower levels of receptor compared to their non-invasive counterparts. These studies indicate a correlation between invasive phenotype and low receptor expression in different tumor cells. Experiments were performed to understand two possible mechanisms (decreased transcription of the receptor and increased transcription of an inhibitory protein) for the decreased cell-surface expression of the receptor in invasive cells. The decreased expression in invasive subclones was due to 2- to 3-fold lower levels of LRP/alpha 2-MR mRNA in all cells. In invasive PC-3 subclones, but not in DU 145, A2058, and MCF-10A subclones, 2-fold higher levels of the 39 kDa receptor-associated protein (an inhibitor of the receptor) mRNA were observed. These studies showed that the decreased expression of LRP/alpha 2-MR activity in invasive subclones was generally correlated with the decreased steady-state mRNA levels of the receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Contribution of vitamin A deficiency to calculogenic risk factors of urine: studies in children.

Studies in experimental animals showed that vitamin A deficiency enhanced the severity of urinary calculi disease. In India, children with low socioeconomic status are the major victims of bladder stone disease, and vitamin A deficiency is also more prevalent among these children. However, no systematic study is available to correlate the vitamin A-deficient status of children with their predisposition to urinary calculi disease. Vitamin A-deficient and normal boys were the subjects of this study. Twenty-four-hour samples of urine were collected from all the children at the beginning of the study and after normalizing the vitamin A status of the deficient children. Important risk factors were estimated in urine. Plasma vitamin A levels were also measured in these children. Among the deficient group, only children with plasma vitamin A levels of 15 micrograms and lower exhibited calcium oxalate crystalluria. Most importantly, abnormal crystalluria was observed in all children whose plasma vitamin A levels were 13 micrograms/dl or less. Compared to normal children the urine of vitamin A-deficient children showed the following changes: (a) reduced concentration of crystal growth inhibitors, namely citrate and glycosaminoglycans; (b) a decline in inhibitory activity toward calcium oxalate crystal growth; and (c) enhanced excretion of high risk factors, namely calcium and oxalate. Correction of vitamin A status normalized the above abnormal properties of urine. The results of this study strongly support the hypothesis that the vitamin A-deficient state is one of the factors that can enhance the risk of urolithiasis in susceptible populations.