Receptor Protein Synthesis Research Articles

Regulation of the thrombin receptor response in human endothelial cells

At present only little information is available about the regulation of the thrombin receptor activity in human endothelial cells. The study presented was performed to clarify the problem of thrombin receptor regulation in human endothelial cells. Endothelial cells were isolated from the vein or artery of human umbilical cords. These cells were stimulated with thrombin or with the thrombin receptor activation peptides (TRAP) SFLLRN or SFLLRNPNDKYEPF and subsequently the von Willebrand factor (vWf) release was measured as a physiologically significant response regarding the thrombin receptor activation. Shortly after the first activation by thrombin or SFLLRN, the cells were desensitised to a second stimulation. After a recovery phase of 10 min, merely 35% of the receptor response was obtained by subsequent stimulation. Expanding the recovery time to 90 min resulted in a vWf release of nearly 100% of the amount measured after the first stimulation. The resensitisation rate was similar for thrombin and SFLLRN stimulated cells. Adding RNA synthesis inhibitors or protein synthesis inhibitors had no effect on the recovery of the receptor response. Therefore, a de novo synthesis of receptor protein must be excluded as a means of resensitising endothelial cells to thrombin. It was shown that the dephosphorylation of tyrosine kinase inactivated receptors is also not responsible for receptor regeneration. These results prove that endothelial cells are capable of developing full thrombin receptor activity after a relatively short recovery phase of only 60–90 min as compared to the recovery phase of 16–24 h in megacaryoblastic cell lines. Desensitisation is similar in thrombin and TRAP stimulated cells. We assumed a transport mechanism for intracellular stored vesicles for receptor resensitisation; a co-localisation with vWf in the Weibel-Palade bodies is improbable.

Inhibitors of Cholesterol Biosynthesis Increase Hepatic Low-Density Lipoprotein Receptor Protein Degradation

Inhibitors of cholesterol biosynthesis are believed to lower serum cholesterol levels by enhancing the removal of serum low-density lipoprotein (LDL) by increasing hepatic LDL receptor function. Thus, the effects of several different inhibitors of cholesterol biosynthesis were examined for their effects on the expression of the hepatic LDL receptor in rats. We found that administration of inhibitors of 3- hydroxy-3-methylglutaryl–coenzyme A reductase such as lovastatin, pravastatin, fluvastatin, and rivastatin resulted in increased hepatic LDL receptor mRNA levels. Surprisingly, these agents failed to increase levels of immunoreactive LDL receptor protein in rat liver even when the dose and length of treatment were increased. Treatment of rats with zaragozic acid A, an inhibitor of squalene synthase, caused even greater increases in hepatic LDL receptor mRNA levels, but did not increase levels of immunoreactive protein. Further investigation revealed that the rate of degradation of the hepatic LDL receptor was increased in rats given inhibitors of cholesterol biosynthesis. The greatest increase in the rate of degradation was seen in animals treated with zaragozic acid A which caused the largest increase in hepatic LDL receptor mRNA levels. In contrast, hepatic LDL receptor protein was stabilized in cholesterol-fed rats. It appears that increased potential for LDL receptor protein synthesis, reflected in increased mRNA levels, is offset by a corresponding increase in the rate of receptor protein degradation resulting in constant steady-state levels of hepatic LDL receptor protein. These findings are suggestive of increased cycling of the hepatic LDL receptor. This postulated mechanism can provide for enhanced hepatic uptake of lipoproteins without increasing steady-state levels of LDL receptor protein.

Chronic alcoholization alters the expression of 5-HT 1A and 5-HT 1B receptor subtypes in rat brain

The expression of central 5-HT 1A and 5-HT 1B receptors was studied in several brain areas of rats subjected to a 2-week period of chronic alcoholization, followed by 18 h withdrawal. Quantitative autoradiography indicated that the ethanol treatment provoked an increase (∼ + 30%) in the labeling by [ 3H]8-hydroxy-2-(di- n-propylamino)tetralin ([ 3H]8-OH-DPAT) and [ 3H] N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]- N-(2-pyridinyl) cyclohexane carboxamide ([ 3H]WAY-100635) of 5-HT 1A autoreceptors in the dorsal raphe nucleus, accompanied by a concomitant decrease in the labeling of postsynaptic 5-HT 1A receptors in the hippocampus (∼ −20%), anterior (∼ −30%) and posterior (∼ −32%) cortices. These changes were associated with a tendency toward an increase and decrease in 5-HT 1A mRNA levels in the anterior raphe area and hippocampus, respectively, suggesting that the changes observed are due to modifications in 5-HT 1A receptor protein synthesis. The autoradiographic labeling of 5-HT 1B receptors by serotonin- O-carboxymethylglycyl[ 125I]iodotyrosinamide ([ 125I]GTI) was found to increase (+55%) in the globus pallidus of alcoholized rats. Interestingly, a significant increase (+57%) in 5-HT 1B receptor mRNA levels was observed in the striatum, which contains cell bodies of neurons projecting into the globus pallidus. These data suggest that altered sensitivity of chronically alcoholized rats to 5-HT 1A and 5-HT 1B receptor ligands may result from alcohol-induced changes in the transcription of the genes encoding these receptors.

IL-13 induces IL-1 receptor antagonist gene expression and protein synthesis in human blood mononuclear cells: Inhibition by an IL-4 mutant protein: A. Salazar-Montes, E. Vannier, R. de Waal Malefyt ∗ and C.A. Dinarello. Tufts University School Medicine, Boston, MA 02111 and ∗DNAX, Palo Alto, CA 94304

IL-13 induces IL-1 receptor antagonist gene expression and protein synthesis in human blood mononuclear cells: Inhibition by an IL-4 mutant protein: A. Salazar-Montes, E. Vannier, R. de Waal Malefyt ∗ and C.A. Dinarello. Tufts University School Medicine, Boston, MA 02111 and ∗DNAX, Palo Alto, CA 94304

Neoglycosylated liposomes as efficient ligands for the evaluation of specific sugar receptors on macrophages in health and in experimental leishmaniasis.

Receptors interacting with terminal sugars as ligands are involved in the binding of Leishmania donovani promastigotes to the macrophage surface and their subsequent internalization. Mannose and glucose are specifically involved in the binding process. Decreased binding occurs to macrophages already infected with L. donovani either in vivo or in vitro. When mannose- or glucose-bearing liposomes are used as ligands the binding shows similar trends and the percentage inhibition of binding with mannose-bearing liposomes increases when compared to that for the glucose-bearing ones. The decreased binding of the ligand seems to be due to a decrease in the number of receptors after infection. The affinity of the ligands for the binding sites either on the normal macrophages or on the infected macrophages apparently remains the same. The results based on the incorporation of [3H]phenyl alanine and supported by the binding of glycosylated liposomes to both infected and non-infected macrophages suggest that protein synthesis, in general, is suppressed in L. donovani-infected macrophages thus affecting also mannose/glucose receptor protein synthesis, resulting in fewer receptors on the macrophage surface.

Increased low density lipoprotein receptor expression mediated through the insulin-like growth factor-I receptor in cultured fibroblasts.

Plasma insulin-like growth factor-I (IGF-I) levels are inversely correlated with apolipoprotein B and low density lipoprotein (LDL) cholesterol in humans. To identify a molecular basis for this observation, the effects of IGF-I on LDL receptor expression in fibroblasts were studied. IGF-I increased LDL receptors in cultured human skin fibroblasts at concentrations greater than 25 ng/ml. However, IGF-I effects were not easily quantitated due to secretion of inhibitory IGF-binding proteins by the cells. To circumvent this difficulty, QAYL, an IGF-I analog that binds to the IGF-I receptor but not to IGF-binding proteins, was used. QAYL increased LDL receptor number 56-72% with half-maximum effect at 0.6 ng/ml. alpha-IR3, a monoclonal antibody directed toward the IGF-I receptor, blocked this effect. QAYL treatment increased synthesis of LDL receptor protein without increasing LDL receptor mRNA levels or altering protein stability. Both QAYL and IGF-I increased LDL receptors prominently in cells that had been treated with physiological amounts of LDL cholesterol. IGF-I, acting through the IGF-I receptor and modulated by IGF-binding proteins, may contribute to the regulation of LDL metabolism by increasing translation of LDL receptor message.

Transient elevation of cAMP by prostaglandins triggers subsequent up-regulation of LDL receptor activity in cultured human cells

Previous work [ 1.2.31 in this laboratory has shown that increased cell cAMP has dual effects on expression of the LDL receptor (LDLR) in human exlrahepatk cells (hSF and hVSMC). i) CAMP upregulates JDLR activity by increasing the rate of receptor protein synthesis; this is not secondary to sterol regulation as the effect persists in the presence of 25-hydroxycholesterol and is unaffected by the HMGCoA reductase inhibitor mevinolin. ii) CAMP also stimulates lysosomal cholesterol ester hydrolase [4], releasing free cholesterol which can inhibit LDLR expression. We have shown 151 that PGE1, E2 and the stable PGI2 analogue Iloprost increase LDLR activity in hSF and hVSMC. This is a true up-regulation, requiring at least 8h preincubation. and depending on mRNA and protein synthesis. We now consider the mechanism of this PG-mediated upregulation. To simplify interpretation of the data, we carried out the investigations in the presence of the lysosomal inhibitors CQ or WCl to prevent down-regulation of the LDLR due to mobilization of cholesterol from lysosomal esters as explained in ii) (above). To show that PG caused real up-regulation of the LDLR, all experiments were carried out on cells in which LDLR number was already up-regulated by 24h preincubation in LPDM. Cells were then incubated with P G s and lysosomal inhibitors for up to 24h in SFM before removing these agents for LDLR measurements. Cell surface expression of the LDLR was measured by the specific binding of [lZr]LDL at 4OC [l]. Cell CAMP was assayed according to [6] m d expressed as % of acid soluble &nine n:icltmtides. While it is well established that PGs can act through elevation of CAMP, the mechanism can differ according to cell type. Accordingly we determined whether PG receptors were functionally linked to adenylate cyclase in the cells used for our LDLR measurements. Forskolin, a direct activator of AC. was used as a positive control. cAMP content was measured after l h with each PG. Table 1 shows that those PG which elevate LDLR activity after 24h also increase cell cAMP content (measured after lh). P G s which were without effect on CAMP content did not affect LDLR expression.

Pubertal maturation is associated with an increase in the number of androgen receptor-immunoreactive cells in the brains of male ferrets

Androgen receptor-immunoreactive (AR-IR) cells were identified in brains of male ferrets before and after the onset of pubertal maturation. There was a greater number of AR-IR cells after the onset of pubertal maturation in some, but not all, brain regions examined. Regions in which the number of AR-IR cells increased included the preoptic area and the amygdala, areas known to be involved in the control of male reproductive behaviors. The mechanisms responsible for the increase in AR-IR cells are unknown, but might be related to the higher circulating levels of testosterone that were present in the older animals. Testosterone may increase androgen receptor (AR) immunoreactivity both by concentrating already existing ARs within the nucleus and by stimulating de novo synthesis of receptor protein.

Potentiation by cholera toxin of bradykinin-induced inositol phosphate production in the osteoblast-like cell line MC3T3-E1.

Cells of the osteoblastic cell line MC3T3-E1 were shown to contain at least three phosphatidylinositol-specific phospholipase C (PI-PLC) isoenzymes (PLC-beta, PLC-gamma and PLC-delta) by Western blotting analysis with various anti-PLC antibodies. Stimulation of inositol phosphate production in MC3T3-E1 cells by bradykinin (BK) occurred via a GTP-binding protein. Inositol phosphate formation on stimulation by BK was not affected by pretreatment with pertussis toxin, whereas it was potentiated by cholera toxin pretreatment. Elevation of cellular cyclic AMP levels by brief pretreatment with dibutyryl cyclic AMP or forskolin failed to enhance the BK-mediated generation of inositol phosphates, but long-term preincubation with these agents partially mimicked the action of the cholera toxin. Cholera toxin also caused an increase in BK receptor number. Cycloheximide, a protein biosynthesis inhibitor, prevented the potentiating actions of the cholera toxin and the cyclic AMP-elevating agents on BK-induced inositol phosphate production, and also inhibited the increase in BK receptor number. The specific binding of [3H]BK to the whole MC3T3-E1 cells in the presence or absence of cholera toxin was completely inhibited by the B2 BK receptor antagonist D-Arg[Hyp3,Thi5,8,D-Phe7]BK, but not by the B1 BK receptor agonist des-Arg9-BK. These data suggest that the activation of PI-PLC induced by cholera toxin in BK-stimulated MC3T3-E1 cells was caused by an enhancement of the synthesis of BK receptor protein(s), at least part of which was mediated by a sustained increase in the intracellular level of cyclic AMP.

Cyclic AMP-mediated hormonal control of LDL receptor activity in cultured human cells

Previous work in this laboratory [ 1.2.31 showed that elevation of cyclic AMP has dual effects (A and B below) on the expression of the LDL receptor in human vascular smooth muscle cells (VSMC) and skin fibroblasts. A). Cyclic AMP elevating agents stimulate LDL receptor activity [ I ] in human cells by increasing the rate of receptor protein synthesis [2]. The stimulation is not secondary to the decrease of the regulatory pool of free cholesterol since it is unaffected, or even enhanced, by inhibition of cholesterol synthesis and esterification, or by inhibition of the conversion of cholesterol to its repressor metabolites [2]. The cyclic AMP mediated up-regulation of the receptor persists in the presence of repressors such as LDL and 25hydroxycholesterol but does not ovemde their effects [2]. B). Cyclic AMP-elevating agents also stimulate the hydrolysis of lysosomal cholesterol esters [4], thus increasing the cellular cholesterol pool and repressing the expression of the LDL receptor. This cholesterol-mediated repressive effect of cyclic AMP can be prevented by chloroquine (CQ) and m C 1 which inhibit lysosomal actions or by ketoconazole which inhibits conversion of free cholesterol to its repressor metabolite [2,3]. It is well established that the action of some prostaglandins are mediated through elevation of cyclic AMP. Here we report the effects of prostaglandins(PG) El, E2 and the stable PGI2 analogue, Iloprost, on regulation of the LDL receptor. In order to simplify interpretation of the data, we have carried out the investigations in the presence of the lysosomal inhibitors CQ and N h C l to prevent any down-regulation of the LDL receptor due to mobilization of cholesterol from lysosomal esters as explained in B (above). To show that PG caused real up-regulation of the LDL receptor, all experiments were carried out on cells in which the LDL receptor number was already maximally expressed by preincubation in LPDM for 24h. Cells were then incubated with PGs and lysosomal inhibitors for up to 24h before removing these agents for LDL receptor measurements. Cell surface expression of the LDL receptor was measured by the specific binding of [1251]LDL at 4OC [l]. Metabolism of LDL by the receptor pathway was determined (after removal of m C I ) by hydrolysis of LDL-borne cholesterylesters to give intracellular free cholesterol. This was assessed by its availability for esterification with [3H]oleate [3]. In fibroblasts, 24h preincubation with PGEl caused significant, concentration-related elevation of LDL binding; 2.8pM PGEl raised binding by 4 3 5 % (PcO.Ol), 14pM PGEl gave a 209f46% (Pd.01) increase above controls. PGE2 yielded similar results, but higher doses (14pM) were needed to ensure consistent effects. In VSMC, 2.8pM Iloprost gave a 5W7C (P4.002) elevation in LDL binding, rising to 128+12% (P<O.OOl) at 14pM. Iloprost effects on fibroblasts showed similar trends. LDL metabolism in VSMC was similarly stimulated by all 3 PGs. At 2.8pM, PGEl gave a 99f16% (P<O.Ol) increase, W E 2 gave a 55f3% (Pc0.02) increase and Iloprost gave a 75+4% (P<O.OOI) increase. PGEl was shown to have no influence on the esterification process itself, all effects being mediated through supply of free cholesterol via up-regulation of the LDL receptor. Study of the timecourse of action of Iloprost on LDL binding showed a marked time dependency with no significant stimulation being observed until after 8h with the agent (Fig. 1). There was a small, but significant, inhibition of binding at 4h.

Regulation of the surface expression of the platelet-activating factor receptor in IC-21 peritoneal macrophages. Effects of lipopolysaccharide.

The effect of bacterial lipopolysaccharide (LPS) on the expression of the receptor for platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; AGEPC) was examined in cultured IC-21 peritoneal macrophages. AGEPC binding to its receptors reached saturation within 20 min at 25 degrees C and was reversible. Scatchard analysis revealed a single class of AGEPC receptors with a Bmax of approximately 170 fmol/mg cellular protein and a Kd of 0.25 nM. Preincubation of IC-21 cells with LPS (0.01-1,000 ng/ml) induced an increase in the surface expression of AGEPC receptors in a time- and concentration-dependent fashion. The maximal effect of LPS on the AGEPC receptor was observed between 5 and 8 h, with a typical increase between 150 and 200%. Scatchard analysis indicated that LPS treatment of IC-21 cells increased the number of AGEPC receptors on the cell surface without any apparent change in the affinity of the receptor for the ligand. The effect of LPS on the surface expression of the AGEPC receptor was nearly abolished by cycloheximide (0.1 mM) and by actinomycin D (3 microM), suggesting the involvement of enhanced receptor protein synthesis and mRNA production in this event. Moreover, LPS treatment increased the capability of the IC-21 cell to respond to AGEPC addition by elevating intracellular free Ca2+ without causing an increase in the basal level of intracellular Ca2+. The present study demonstrates that IC-21 peritoneal macrophages possess high affinity AGEPC receptors and provides the evidence that the number of functional AGEPC receptors on a cell can be increased significantly upon exposure to LPS.

Enhanced epidermal growth factor receptor synthesis in human squamous carcinoma cells exposed to low levels of oxygen.

Exposure of human A431 squamous carcinoma cells to levels of hypoxia found in some solid tumors causes 2-fold increases in epidermal growth-factor receptor (EGF-R) mRNA levels and rate of receptor protein synthesis compared with aerobic cells. Similar results are shown for receptor message from other squamous carcinoma cells, human keratinocytes, and human W138 fibroblasts. Less basal tyrosine phosphorylation of the receptor occurs in hypoxic compared with aerobic A431 cells. Scatchard analysis also shows that reoxygenated A431 cells display enhanced surface expression of the EGF-R compared with aerobic control cells. Possible mechanisms and implications for tumor therapy are discussed.

Dexamethasone up-regulates mannose receptor activity by increasing mRNA levels

The macrophage mannose receptor is highly susceptible to modulation by a variety of inflammatory and anti-inflammatory agents. Previous studies have demonstrated that mannose receptor activity is dramatically enhanced in rat bone marrow macrophages following treatment with dexamethasone. In the present study we have investigated potential mechanisms that might be involved in this up-regulation. Uptake of ligands by the mannose receptor was increased 2.5-fold in a dose- and time-dependent manner. Maximal stimulation was seen following treatment of macrophages with 0.1–1.0 μg/ml of dexamethasone for 24–48 h. Dexamethasone treatment increased both the number of cell surface binding sites and total cellular binding activity to 250% of control levels. In addition, total receptor protein as measured by immunoprecipitation was increased 2.5-fold. Neither the maturation rate nor the turnover rate of the protein was altered by dexamethasone treatment. Using an oligonucleotide probe derived from sequence data from the cloned human receptor cDNA, we investigated the effect of dexamethasone on the expression of mannose receptor mRNA. Following incubation with dexamethasone for 12–24 h, the level of mRNA was significantly increased. These results demonstrate that dexamethasone treatment of rat bone marrow macrophages induces synthesis of new receptor protein through an increase in the level of mannose receptor mRNA.

Regulation of LDL receptor, apoB, and apoE protein and mRNA in Hep G2 cells.

The regulation of low-density lipoprotein (LDL) receptor activity, protein synthesis, and cellular mRNA content was evaluated in the human hepatoma cell line Hep G2. Incubation of the cells with LDL led to a complete downregulation of LDL receptor mRNA and LDL receptor protein synthesis. This LDL regulation of the LDL receptor and its mRNA was both time- and concentration-dependent. In contrast to protein synthesis and cellular mRNA concentrations of the LDL receptor, which were reduced to undetectable levels by prolonged incubation in the presence of LDL, LDL receptor activity was reduced to only 44% of preincubation levels. These findings support the presence of a second metabolic pathway for LDL uptake in human hepatocytic cells. The effect of LDL on cellular LDL receptor expression was specific for LDL because incubation in the presence of HDL did not affect any of these study end points. The potential coordinate regulation of the expression of the LDL receptor with its principal ligands, apolipoproteins (apo) B and E, was also investigated. In contrast to the LDL receptor mRNA downregulation with LDL incubation, cellular apoB and apoE mRNA concentrations were not affected by either LDL or HDL. Secretion of apoB, however, was significantly increased by incubating Hep G2 cells with LDL. These findings indicate that, in contrast to LDL receptor which is regulated at the mRNA level, the ligands for the LDL receptor are regulated either co- or post-translationally.

Effects of bicuculline on [3H]SR 95531 binding in discrete regions of rat brains.

Effects of bicuculline in vitro, and acute and chronic treatment of a subconvulsive dose of bicuculline on [3H]SR 95531 binding to discrete regions of rat brains were studied in Sprague-Dawley rats. Scatchard analysis of the binding isotherms exhibited two populations of binding sites for [3H]SR 95531 in frontal cortex, cerebellum, striatum and substantia nigra. The apparent KD for high-affinity sites was significantly increased in the frontal cortex and cerebellum in the presence of bicuculline (1 microM) with no change in Bmax. In contrast, the apparent affinity for low-affinity sites was not altered in the presence of bicuculline in these regions, whereas the Bmax was significantly decreased in the cerebellum. Following acute (2 mg/kg, i.p.) or chronic (2 mg/kg, i.p. for 10 days) bicuculline treatment, [3H]SR 95531 binding was also investigated in various regions of brains. The acute bicuculline treatment did not affect the [3H]SR 95531 binding in any of the regions studied. In contrast, apparent affinity for [3H]SR 95531 was significantly decreased in low-affinity sites of all regions studied in rats treated chronically with bicuculline. The Bmax values of high and low-affinity sites were significantly increased in the cerebellum with no change in the frontal cortex, striatum and substantia nigra. The present study demonstrates that chronic bicuculline treatment decreases apparent affinity of [3H]SR 95531 binding whereas the treatment increases apparent affinity of [3H]muscimol binding (1) in various brain regions. The results indicate that significant increase in Bmax of [3H]SR 95531 and [3H]muscimol binding in the cerebellum may be due to true up-regulation of GABA binding sites, involving increased de novo synthesis of receptor protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic AMP stimulates the synthesis and function of the low-density lipoprotein receptor in human vascular smooth-muscle cells and fibroblasts.

1. Cyclic AMP-elevating agents stimulate low-density lipoprotein (LDL) receptor activity in human vascular smooth-muscle cells by increasing the rate of receptor protein synthesis. The stimulation is not secondary to the decrease in the regulatory pool of free cholesterol, since it is unaffected, or even enhanced, by inhibition of cholesterol synthesis and esterification, or inhibition of the conversion of cholesterol into its repressor metabolites. The cyclic AMP-mediated up-regulation of the receptor is maintained at low concentrations of inhibitory sterols, but is eventually over-ridden at high concentrations of these sterols. 2. Cyclic AMP-elevating agents also stimulate the hydrolysis of lysosomal cholesterol esters, thus increasing the cellular cholesterol pool and repressing the expression of the LDL receptor. This cholesterol-mediated repressive effect of cyclic AMP can be prevented by chloroquine, which inhibits lysosomal actions, or by ketoconazole, which inhibits conversion of free cholesterol into its repressor metabolite. Thus the cyclic AMP stimulation of the LDL receptor can be masked by the rapid mobilization of free cholesterol from existing cholesterol esters within cultured cells. 3. We have observed that elevated cyclic AMP concentrations will up-regulate the LDL receptor in cholesterol-depleted human vascular smooth-muscle cells, skin fibroblasts and foetal-lung fibroblasts. We propose that our results are evidence for a cyclic AMP-stimulated, sterol-independent, control of LDL-receptor synthesis which is of widespread occurrence in human cells.

Dexamethasone and retinoic acid regulate the expression of epidermal growth factor receptor mRNA by distinct mechanisms

Retinoic acid and dexamethasone have antagonistic effects on epidermal growth factor (EGF) receptor expression in fetal rat lung (FRL) cells: Receptor synthesis is enhanced by retinoic acid and reduced by dexamethasone. In the presence of actinomycin D, neither agent has the capacity to modify receptor synthesis or 125I-EGF binding capacity. Northern blot analysis demonstrates a tenfold increase in EGF mRNA following retinoic acid treatment and a 60% decrease in receptor message levels after dexamethasone treatment. To dissect the mechanisms of these effects, the expression of mRNA was separated from effects requiring protein synthesis by the use of cycloheximide and actinomycin D. Ligand binding, EGF receptor protein synthesis, and mRNA levels were measured in cultures of FRL cells that were incubated with retinoic acid or dexamethasone in the presence of cycloheximide, then washed and reincubated with fresh media containing actinomycin D, but not retinoic acid, dexamethasone, or cycloheximide. The results demonstrate that dexamethasone reduces the expression of EGF receptor mRNA in the absence of protein synthesis. In contrast, the mechanism by which retinoic acid increases the expression of EGF receptor mRNA requires protein synthesis. These data indicate that, in FRL cells, dexamethasone negatively regulates EGF receptor mRNA in a direct manner, while retinoic acid controls transcription of an intermediate protein, possibly a transcription factor, that subsequently increases transcription of receptor message.

Inactivation of a sperm motility gene by insertion of an epidermal growth factor receptor transgene whose product is overexpressed and compartmentalized during spermatogenesis.

Transgenic mice were generated with a human epidermal growth factor (EGF) receptor cDNA driven by the chicken beta-actin gene promoter. One line (AE24) that exhibited a unique expression pattern in which dramatically elevated levels of EGF receptor RNA were found only in the testis was established, suggesting that the beta-actin promoter was being influenced by an adjacent testis-specific enhancer. EGF receptor RNA was detected in primary spermatocytes, whereas the synthesis of receptor protein was restricted to elongate spermatids, indicating that transgene expression was under translational control. At spermiation, the EGF receptor was sequestered in residual bodies and excluded from mature sperm by a compartmentalization mechanism. About half of AE24 homozygous males were sterile because of sperm paralysis, whereas heterozygous males and females of either genotype were completely fertile. Electron microscopic analysis of sperm flagella from sterile AE24 homozygotes revealed an aberrant axonemal structure in which outer doublet microtubules were missing from the middle piece, resembling changes observed in the sperm of some infertile humans. Flagellar axonemal disassembly was observed in the vas deferens and epididymis but not in the testis, suggesting that outer doublets were assembled in a grossly normal manner but possessed a latent instability. These results demonstrate that in the AE24 mouse line the EGF receptor transgene was integrated into and inactivated an endogenous autosomal gene, causing sperm flagellar axonemal disruption and male sterility.

Changes in serum lysosomal enzymes in the vitamin B 6-deficient weanling rat

The goal of this study was to investigate the effect of vitamin B 6 deficiency on the level of a variety of serum lysosomal hydrolases in the rat. Twentyone day old weanling, male Holtzman rats were divided into three groups. Group I was fed ad libitum a diet adequate in vitamin B 6; Group II was fed ad libitum a diet deficient in vitamin B 6; animals in Group III were matched with Group II animals and pair-fed the control diet. Animals in Group II exhibited evidence of a vitamin B 6-deficient state: they gained weight at less than half the rate of those in Group I and their serum was characterized by markedly decreased alanine aminotransferase and aspartate aminotransferase activities. The activities of a number of serum lysosomal hydrolases were determined at 11, 25, and 47 days following weaning. By the 47th day, with the exception of acid phosphatase activity (which remained unchanged during the course of the experiment), the activities of the following enzymes were increased 1.5–3.0 fold: β-galactosidase, β-glucuronidase, α-fucosidase, and the acid (pH 4.2) and intermediate (pH 5.5) α-mannosidase. Fractionation of sera on DEAE-Sephadex columns prior to assay indicated that the increase in intermediate pH optimum α-mannosidase activity was attributable to an elevation in the activity of only one of the two principal intermediate pH optimum isoenzymes present in rat serum. Although the molecular explanation for the observed alterations in serum lysosomal hydrolases is not apparent, the possibility is discussed that vitamin B 6 could play some critical role in the targeting of lysosomal proteins to that organelle. Specifically, the vitamin in its cofactor form may participate in the synthesis of mannose phosphate marker or receptor protein on lysosomes which dictate the disposition of these proteins.

Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA

Human umbilical vein endothelial cells in culture (HUVEC) express receptors for urokinase-type plasminogen activators (u-PA). The immunochemical nature of this receptor and its relationship to u-PA receptors expressed by other cell types is unknown. Cross-linking active site-blocked u-PA to HUVEC lead to an increase in its apparent molecular mass by approximately 40 Kd. The predominant u-PA binding protein isolated from whole cell detergent extracts migrated with a molecular mass of approximately 36 Kd using affinity chromatography. In contrast, when only cell surface proteins were radiolabeled before extraction, the predominant labeled u-PA binding protein isolated migrated with a molecular mass of approximately 46 Kd. Several pieces of evidence suggested that the difference in molecular mass between these two u-PA binding proteins resulted from glycosylation of a single receptor protein. First, a polyclonal antibody against u-PA receptor isolated from phorbol myristate acetate (PMA) stimulated U-937 cells reacted with both the 36- and 46-Kd proteins on Western blotting. Second, the size of the unmodified receptor was estimated by amplifying a full-length cDNA for u-PA receptor from an endothelial cell cDNA library using the polymerase chain reaction (PCR) and oligonucleotide primers corresponding to the DNA sequence of the receptor cloned from transformed human fibroblasts (Roldan et al, EMBO J 9:467, 1990). The size of the cDNA (approximately 1,054 base pairs, bp) and the presence of a single 1.4-kilobase (Kb) mRNA transcript on Northern blot analysis predict an unglycosylated receptor protein of approximately 35 Kd. Third, synthesis of 35S-labeled 46-Kd cell surface receptor protein was inhibited when the cells were grown in the presence of tunicamycin, while the synthesis of the 36-Kd species was unaffected. Moreover, the apparent molecular mass of purified surface-labeled receptor (approximately 46 Kd) was reduced by N-glycanase. These studies suggest that the u-PA receptor on the surface of HUVEC is a glycoprotein derived from a protein of approximately 35 Kd which is similar immunologically to u-PA receptors on other cell types.