Receptors In Human Fibroblasts Research Articles

ATM is necessary for IGF-1R expression in the murine mammary gland

Ataxia-telangiectasia mutated is a protein kinase that is critical for the maintenance of genomic stability and proper cellular response to redox imbalance. Previous studies indicate that ataxia-telangiectasia mutated is also required for expression of the insulin-like growth factor-1 receptor in human fibroblasts. As insulin-like growth factor-1 receptor is critical in mammary gland development, we sought to determine the relationship between ataxia-telangiectasia mutated and insulin-like growth factor-1 receptor using mouse as a model system. Knockdown of ataxia-telangiectasia mutated in cultured mouse mammary epithelial cells resulted in a significant reduction in insulin-like growth factor-1 receptor mRNA levels. Using a conditional knockout mouse model, we observed that loss of ataxia-telangiectasia mutated resulted in a severe decrease in both insulin-like growth factor-1 receptor mRNA and protein expression. These results support the observation that insulin-like growth factor-1 receptor expression is ataxia-telangiectasia mutated -dependent in the mammary epithelium, and given the critical role that insulin-like growth factor-1 receptor plays in mammary gland development, suggests that ataxia-telangiectasia mutated may be critical in the development of this organ as well.

Surface aggregation patterns of LDL receptors near coated pits III: potential effects of combined retrograde membrane flow-diffusion and a polarized-insertion mechanism

Although the process of endocytosis of the low density lipoprotein (LDL) macromolecule and its receptor have been the subject of intense experimental research and modeling, there are still conflicting hypotheses and even conflicting data regarding the way receptors are transported to coated pits, the manner by which receptors are inserted before they aggregate in coated pits, and the display of receptors on the cell surface. At first it was considered that LDL receptors in human fibroblasts are inserted at random locations and then transported by diffusion toward coated pits. But experiments have not ruled out the possibility that the true rate of accumulation of LDL receptors in coated pits might be faster than predicted on the basis of pure diffusion and uniform reinsertion over the entire cell surface. It has been claimed that recycled LDL receptors are inserted preferentially in regions where coated pits form, with display occurring predominantly as groups of loosely associated units. Another mechanism that has been proposed by experimental cell biologists which might affect the accumulation of receptors in coated pits is a retrograde membrane flow. This is essentially linked to a polarized receptor insertion mode and also to the capping phenomenon, characterized by the formation of large patches of proteins that passively flow away from the regions of membrane exocytosis. In this contribution we calculate the mean travel time of LDL receptors to coated pits as determined by the ratio of flow strength to diffusion-coefficient, as well as by polarized-receptor insertion. We also project the resulting display of unbound receptors on the cell membrane. We found forms of polarized insertion that could potentially reduce the mean capture time of LDL receptors by coated pits which is controlled by diffusion and uniform insertion. Our results show that, in spite of its efficiency as a possible device for enhancement of the rate of receptor trapping, polarized insertion nevertheless fails to induce the formation of steady-state clusters of receptor on the cell membrane. Moreover, for appropriate values of the flow strength-diffusion ratio, the predicted steady-state distribution of receptors on the surface was found to be consistent with the phenomenon of capping.

ApoE and Aβ in Alzheimer’s Disease: Accidental Encounters or Partners?

Among the three human apolipoprotein E (apoE) isoforms, apoE4 increases the risk of Alzheimer's disease (AD). While transporting cholesterol is a primary function, apoE also regulates amyloid-β (Aβ) metabolism, aggregation, and deposition. Although earlier work suggests that different affinities of apoE isoforms to Aβ might account for their effects on Aβ clearance, recent studies indicate that apoE also competes with Aβ for cellular uptake through apoE receptors. Thus, several factors probably determine the variable effects apoE has on Aβ. In this Review, we examine biochemical, structural, and functional studies and propose testable models that address the complex mechanisms underlying apoE-Aβ interaction and how apoE4 may increase AD risk and also serve as a target pathway for therapy.

Cytomegalovirus Upregulates Vascular Endothelial Growth Factor and Its Second Cellular Kinase Domain Receptor in Human Fibroblasts

Human cytomegalovirus (HCMV) activation and elevated levels of vascular endothelial growth factor (VEGF) have been found to be associated with transplant rejection. However, information is lacking about whether elevated levels of this multifunctional factor are directly due to viral activation, or if they derive from impurities within the culture supernatant of infected cell cultures. We used purified as well as unpurified viral stocks to infect human fibroblasts in vitro and applied PCR, Western blot, ELISA, and immunofluorescence staining to investigate the expression of VEGF and its receptors. Our data suggest that HCMV infection triggers an early and sustained induction of VEGF and kinase insert domain receptor (KDR) mRNAs, whereas transcript levels of FLT-1 remain unchanged by viral infection. Analysis of the extracellular VEGF and cellular KDR protein expression after infection with purified and unpurified HCMV strains AD169 and TOLEDO showed, in clear contrast to UV-inactivated virus preparations, an increased release of VEGF and KDR proteins. In addition, immunofluorescence revealed that HCMV infection was also accompanied by a profound increase in intracellular VEGF and KDR levels. These results confirm that active HCMV infection is required to induce VEGF and the most important VEGF receptor KDR, and that the upregulation of VEGF and KDR are a direct viral effect and not a secondary effect mediated by inflammatory cytokines within the supernatant. The HCMV-dependent upregulation of VEGF and KDR contributes to the theory that viral-induced immune mediators play a key role in transplant rejection.

O-9: Characterisation of hybrid receptors in human fibroblasts

O-9: Characterisation of hybrid receptors in human fibroblasts

Effects of lipoprotein lipase and statins on cholesterol uptake into heart and skeletal muscle

Regulation of cholesterol metabolism in cultured cells and in the liver is dependent on actions of the LDL receptor. However, nonhepatic tissues have multiple pathways of cholesterol uptake. One possible pathway is mediated by LPL, an enzyme that primarily hydrolyzes plasma triglyceride into fatty acids. In this study, LDL uptake and tissue cholesterol levels in heart and skeletal muscle of wild-type and transgenic mice with alterations in LPL expression were assessed. Overexpression of a myocyte-anchored form of LPL in heart muscle led to increased uptake of LDL and greater heart cholesterol levels. Loss of LDL receptors did not alter LDL uptake into heart or skeletal muscle. To induce LDL receptors, mice were treated with simvastatin. Statin treatment increased LDL receptor expression and LDL uptake by liver and skeletal muscle but not heart muscle. Plasma creatinine phosphokinase as well as muscle mitochondria, cholesterol, and lipid droplet levels were increased in statin-treated mice overexpressing LPL in skeletal muscle. Thus, pathways affecting cholesterol balance in heart and skeletal muscle differ.

Modulation of the expression of peroxisome proliferators-activated receptors in human fibroblasts

To determine the levels of peroxisome proliferators-activated receptors (PPARs) in normal and adhesion fibroblasts, we utilized real-time reverse transcription-polymerase chain reaction to measure messenger RNA (mRNA) levels in fibroblasts from normal peritoneum and adhesions from five patients in both the presence or absence of dichloroacetic acid (DCA) and a cyclooxygenase-2 (COX-2) inhibitor, NS-398. Peroxisome proliferators-activated receptor alpha, PPARbeta, PPARgamma1, and PPARgamma2 mRNA are all present in normal peritoneal and adhesion fibroblasts, and selectively rose in response to hypoxia and either DCA or NS-398.

The Effects of Estradiol on the Expression of Estrogen, Progesterone, Androgen, and Prolactin Receptors in Human Peritoneal Fibroblasts

Previous studies have suggested a possible role of hormonal milieu mediating endometriosis and/or postoperative adhesion development. The objective of this study is to determine the expression of hormone receptors in human fibroblasts from normal peritoneum in response to increasing concentrations of estradiol.

Differential Expression of Estrogen, Progesterone, Androgen, and Prolactin Receptors in Human Fibroblasts Isolated From Normal Peritoneum and Adhesions

Adhesions are believed to contribute to infertility in 40% of infertile couples and are the most common cause of small bowel obstruction. Also abdominal-pelvic pain is attributed to adhesions in a little less of 30% of cases. Our objective is to characterize fibroblasts isolated from normal peritoneum and adhesion tissues of the same patient in their ability to express hormone receptors.

Downregulation of bradykinin B2 receptor in human fibroblasts during prolonged agonist exposure.

Sustained activation of G protein-coupled receptors results in an attenuation of cellular responses, a phenomenon termed desensitization. Whereas mechanisms for rapid desensitization of ligand-receptor-G protein-effector systems are relatively well characterized, much less is known about long-term adaptation processes that occur in the continuous presence of an agonist. Here we have studied the fate of endogenously expressed bradykinin B(2) receptors on human fibroblasts during prolonged agonist treatment. Stimulation with bradykinin for up to 24 h resulted in a 50% reduction of surface binding sites that was paralleled by a similar decrease of total B(2) receptor protein followed by Western blotting using monoclonal antibodies to the B(2) receptor. Whereas B(2) receptor mRNA levels did not change during 24 h of agonist treatment, B(2) receptor de novo synthesis was attenuated by 35-50%, indicating translational control of B(2) receptor levels. Furthermore, the half-life of B(2) receptor protein was shortened by 20-40% as shown by (35)S-labeled pulse-chase and immunoprecipitation experiments. This study demonstrates that bradykinin B(2) receptor expression during long-term agonist treatment is primarily regulated on the (post)translational level, i.e., by attenuation of de novo synthesis and by reduction of receptor stability.

Ligand-induced Phosphorylation/Dephosphorylation of the Endogenous Bradykinin B2 Receptor from Human Fibroblasts

We have studied the ligand-induced phosphorylation/dephosphorylation of the bradykinin B2 receptor endogenously expressed in human HF-15 fibroblasts. An antiserum (AS346) to a synthetic peptide (CRS36), derived from the extreme carboxyl terminus of the human B2 receptor, precipitated the receptor from solubilized membranes of HF-15 cells that had been labeled with [32P]orthophosphate. A low basal level of B2 receptor phosphorylation was found in the absence of a ligand. Stimulation of the cells with the B2 receptor agonists bradykinin, [Lys0,Hyp3]bradykinin, kallidin, and T-kinin resulted in a rapid and efficient phosphorylation of the receptor. The B2 receptor antagonist HOE140 and the B1 receptor agonist des-Arg9-bradykinin failed to induce significant phosphorylation of the B2 receptor. Phosphoamino acid analysis revealed that the B2 receptor is phosphorylated on serine and threonine, but not on tyrosine residues. The ligand-induced phosphorylation of the receptor was concentration-dependent, with an apparent EC50 of 33 nM, and peaked at 1 min after challenge. The kinin-stimulated phosphorylation of the B2 receptor was rapid and transient and paralleled the kinetics of desensitization/resensitization of the receptor as followed by [Ca2+]i release and radioligand binding assay, respectively. The ligand-induced phosphorylation of the B2 receptor was independent of the protein kinase C pathway. In vitro experiments suggest betaARK1 (beta-adrenergic receptor kinase) as a candidate kinase that could mediate the homologous B2 receptor phosphorylation. Inhibitors of protein phosphatases 1 and 2A effectively blocked the dephosphorylation, but did not affect the internalization of the B2 receptor, whereas inhibitors of receptor internalization delayed its dephosphorylation. These finding point to a role of ligand-induced phosphorylation in the desensitization and redistribution of the bradykinin receptor in human fibroblasts.

Stimulatory Effect of PDGF on HMG-CoA Reductase Activity and N-Linked Glycosylation Contributes to Increased Expression of IGF-1 Receptors in Human Fibroblasts

In the present paper, we show that the prereplicative period in platelet-derived growth factor (PDGF)-stimulated human diploid fibroblasts (HDF) includes an early increase in 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity followed by an increased N-linked glycosylation. HMG-CoA reductase inhibitors abolished these events and also prevented progression of cells into S-phase, indicating that mevalonate (MVA)-dependent glycosylation might be required in PDGF-mediated cell growth. Furthermore, PDGF was demonstrated to increase the number of insulin-like growth factor 1 (IGF-1) binding sites in HDF. This event, which was dependent on MVA, took place late in the prereplicative period and was correlated to a significant increase in the expression ofde novosynthesized IGF-1 receptor (IGF-1R) proteins at the cell membrane. The PDGF-induced IGF-1R expression was suppressed in the presence of tunicamycin, an inhibitor of N-linked glycosylation. Taken together, our findings suggest an important role of MVA and N-linked glycosylation in PDGF-mediated growth activation of HDF.

The Growth-Inhibitory Block of TGF-β Is Located Close to the G1/S Border in the Cell Cycle

Transforming growth factor-β (TGF-β) inhibits DNA synthesis in dense cultures of young human embryonic fibroblasts and antagonizes the mitogenic action of platelet-derived growth factor (PDGF). The inhibition of the PDGF-BB action by TGF-β was independent of the induction of mRNAs for the PDGF-A chain and PDGF-β receptor, the predominant types of PDGF receptor in human fibroblasts. The TGF-β-mediated inhibition did not influence the expression of various genes that are involved in the transition from the arrested (G0) state to the S phase of the cell cycle. Indeed, TGF-β upregulated the "early" genes c-myc, c-fos, and jun B and downregulated the growth arrest-specific (gas) genes. These results suggest that the inhibition of DNA synthesis by TGF-β in human fibroblasts is independent of modulation of expression of early and gas genes, placing the TGF-β block comparatively late in the G0 to S transition. In cultures of senescent human fibroblasts TGF-β stimulated DNA synthesis but, nevertheless, had the same effect as in young cells on the expression of PDGF chains and receptor genes, as well as on early and gas genes, with the exception of a significantly lower induction of c-fos.

Affinity cross-linking of bradykinin B 2 receptors in guinea pig ileum membranes

Affinity cross-linking of the bradykinin B 2 receptor was performed using [ 125I-Tyr 8]bradykinin, disulfosuccinimidyl tartrate as linker and crude membranes from guinea pig ileum smooth muscle immobilized on Whatman GF/B glass fiber filters. After SDS (sodium dodecylsulfate)-polyacrylamide gel electrophoresis under reducing conditions one major band of an apparent molecular mass of 66 kDa was obtained. The labeling intensity of this band was diminished in the presence of both unlabeled bradykinin and the bradykinin B 2 receptor selective antagonist Hoe 140 ([D-Arg 0,Hyp 3,Thi 5,D-Tic 7,Oic 8]bradykinin) but not by the bradykinin B 1 receptor agonist des-Arg 9-bradykinin. Under non-reducing conditions this band shifted to a 59 kDa position. Considering the molecular mass of covalently bound [ 125I-Tyr 8]bradykinin (1.2 kDa) the bradykinin B 2 receptor in guinea pig ileum smooth muscle membranes is a 65 kDa protein. Its structure seems to be very similar to, and yet slightly distinct from, the 69 kDa bradykinin B 2 receptor in human fibroblasts recently described (Abd Alla et al., 1993, J. Biol. Chem. 268, 17277) suggesting putative differences between bradykinin B 2 receptors in various tissues.

Processing and characterization of the low density lipoprotein receptor in the human colonic carcinoma cell subclone HT29-18: a potential pathway for delivering therapeutic drugs and genes.

Low density lipoprotein (LDL) processing has been investigated in the subcloned human colonic carcinoma cell line HT29-18. LDL binding at 4 degrees C was a saturable process in relation to time and LDL concentration. The Kd for LDL binding was 11 micrograms/ml. ApoE-free HDL3 or acetylated LDL did not significantly compete with 125I-LDL binding, up to 500 micrograms/ml. 125I-LDL binding was decreased by 70% in HT29-18 cells preincubated for 24 hours in culture medium containing 100 micrograms/ml unlabelled LDL. Ligand blotting studies performed on HT29-18 homogenates using colloidal gold labelled LDL indicated the presence of one autoradiographic band corresponding to an apparent molecular weight of 130 kDa, which is consistent with the previously reported molecular weight of the LDL receptor in human fibroblasts. At 37 degrees C, 125I-LDL was actively internalized by HT29-18 cells and lysosomal degradation occurred as demonstrated by the inhibitory effect of chloroquine. LDL uptake and degradation by HT29-18 cells also resulted in a marked decrease in endogenous sterol synthesis. These data demonstrate that the HT29-18 human cancerous intestinal cells are able to specifically bind and internalize LDL, and that LDL processing results in down-regulation of sterol biosynthesis. Thus, intestinal epithelial cells possess specific LDL receptors that can be exploited to accomplish drug delivery and gene transfer via the receptor-mediated endocytosis pathway.

Insulin-like growth factors (IGFs) reduce IGF-binding protein-4 (IGFBP- 4) concentration and stimulate IGFBP-3 independently of IGF receptors in human fibroblasts and epidermal cells

Insulin-like growth factors (IGFs) reduce IGF-binding protein-4 (IGFBP- 4) concentration and stimulate IGFBP-3 independently of IGF receptors in human fibroblasts and epidermal cells

Insulin-like growth factors (IGFs) reduce IGF-binding protein-4 (IGFBP-4) concentration and stimulate IGFBP-3 independently of IGF receptors in human fibroblasts and epidermal cells.

Recent studies have provided a consensus that insulin-like growth factor-I (IGF-I) stimulates IGF-binding protein-3 (IGFBP-3) in vivo and in vitro. While it also appears well established that IGFBP-1 is inversely related to insulin concentrations, evidence regarding regulation of other IGFBP is inconclusive. Using immunoprecipitation and Western ligand blot, we have characterized the IGFBPs released into conditioned medium (CM) by cells from the adult human fibroblast cell line N3652 and the human epidermal squamous cell carcinoma line SCL-1. N3652 cells expressed IGFBP-3, IGFBP-2, a 24-kilodalton (kDa) IGFBP presumed to be IGFBP-4, and IGFBPs at 30 and 28 kDa. SCL-1 expressed IGFBP-3 and a putative IGFBP-4, with intermediate bands at 34 and 30 kDa. As determined by ligand blot of CM from confluent cells 72 h after the addition of peptides to serum-free medium, IGF-I and IGF-II potently stimulated IGFBP-3 in both cell lines, but otherwise IGFBP regulation in the two cells diverged. In N3652 cells, IGFBP-3 concentrations in CM increased to 700% and 800% of basal levels in the presence of IGF-I and IGF-II (at 100 ng/ml; n = 5 experiments), respectively. IGFBP-3 was not affected by insulin up to 10 micrograms/ml. In contrast, IGFBP-4 levels were diminished 54% and 73% by 100 ng/ml IGF-I and IGF-II, respectively, with no response to insulin. In SCL-1 cells, IGF-I and IGF-II were virtually identical in stimulating a mean 200% increase in IGFBP-3 (n = 5 experiments). Insulin was less potent, but caused a significant stimulation of IGFBP-3 levels. IGF-I, IGF-II, and insulin all stimulated an approximately 50% increase in IGFBP-4 concentrations. To test the hypothesis that IGF-induced alterations in IGFBP-3 and IGFBP-4 concentrations were regulated via the type 1 IGF receptor, we attempted to block IGFBP changes with type 1 IGF receptor antibody alpha IR-3 and to induce IGFBP changes with an IGF-II analog, [Leu27]IGF-II, with little affinity for the type 1 receptor. alpha IR-3 failed to block either the IGF-induced rise in IGFBP-3 in each cell line or the decline in IGFBP-4 in N3652 CM. [Leu27]IGF-II was as potent as IGF-II or IGF-I in inducing changes in IGFBP-3 and IGFBP-4 concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin 1 beta induces rapid phosphorylation and redistribution of talin: a possible mechanism for modulation of fibroblast focal adhesion.

The majority of interleukin 1 (IL-1) receptors in human fibroblasts has been shown to be localized at focal adhesions. This study describes rapid alterations caused by IL-1 beta/IL-1-receptor interaction at these sites. Fibroblast monolayers, incubated with IL-1 beta and prepared for electron microscopy, showed successive loss of cell-substratum contact and fewer and less-pronounced processes. Immunocytochemistry revealed loss and redistribution of the talin staining initially observed after 5-15 min of IL-1 beta incubation. Similarly, the cytoskeleton showed a decrease in staining and a disorganization starting from 15 to 30 min after IL-1 addition, whereas extracellular fibronectin appeared largely unaffected. Prelabeling with [32P]phosphate showed a 2- to 3-fold increase in the level of talin phosphorylation, peaking at 15 min. Phospho amino acid analyses revealed a higher level of serine and threonine phosphorylation. The data suggest that the action of IL-1 beta on fibroblasts may be partially mediated by direct phosphorylation of talin via activation of a protein serine/threonine kinase, leading to changes in transmembrane linkage proteins and the cytoskeleton. Such alterations at focal adhesions may provide a mechanism by which IL-1 can rapidly modulate cell-matrix interactions during inflammation and wound healing.

Lateral diffusion of PDGF beta-receptors in human fibroblasts.

When platelet-derived growth factor (PDGF) binds to its receptors a number of biochemical reactions are elicited in the cell. Several models have been presented for the effects of ligand-induced receptor conformation and aggregation on signal transduction but little is known about the direct effects on receptor diffusion. This study concerns the lateral mobility of PDGF receptors in fibroblasts. It was assessed with fluorescence recovery after photobleaching (FRAP), using rhodaminated receptor antibodies or Fab-fragments of the antibody as ligands. The aims of the investigation were: (a) to compare the lateral mobility of membrane receptors of human fibroblasts labelled with either antibodies against the PDGF receptor or Fab-fragments of the same antibodies, and (b) to study the effects of serum or PDGF on the mobility of the receptors. Human foreskin fibroblasts (AG 1523) were grown on coverslips either under standard or under serum-free conditions yielding "normal" and "starved" cells, respectively. Two parameters of the diffusion were evaluated; the diffusion coefficient (D) and the mobile fraction (R) of the receptors. We found that normal fibroblasts had a smaller diffusion coefficient and a lower mobile fraction compared to starved cells using antibodies for receptor labelling. The addition of PDGF, just before the measurement, increased the D and R for normal cells, while starved cells, showing higher initial values, displayed slightly reduced values of D and R. After the addition of serum, D increased and R remained low for normal cells, whereas for starved cells both D and R increased to upper limits of 11.0 x 10(-10) cm2s-1 and greater than 90% respectively. In general, the D and R values, both in normal and starved cells, were higher for cells labelled with Fab-fragments than for antibody-labelled cells. The results are discussed in relation to the natural complexity of the receptor, and how PDGF, serum, antibodies and Fab-fragments might interfere with receptor structure, aggregation state and membrane diffusion characteristics.

Histamine receptors in human fibroblasts: inositol phosphates, Ca2+, and cell growth

Histamine stimulated inositol phosphate formation by human skin fibroblasts. The effect of histamine was reduced but still readily apparent in the absence of extracellular Ca2+. Histamine caused a transient increase in intracellular free Ca2+ as detected by indo-1 and fura-2 fluorescence studies on cell populations and on individual cells. Similar increases were observed in the absence of extracellular Ca2+, indicating that the effect was primarily due to mobilization of Ca2+ from intracellular stores, presumably by inositol trisphosphate (IP3). The effects of histamine on phosphoinositide metabolism and intracellular Ca2+ were inhibited by pretreatment of the cells with phorbol esters, suggesting that the histamine receptor in fibroblasts is subject to feedback regulation by protein kinase C. Histamine inhibited the incorporation of [3H]-thymidine into DNA. The effects of histamine on inositol phosphate formation, intracellular Ca2+, and thymidine incorporation were blocked by the H1 receptor antagonist mepyramine. Our results indicate that human skin fibroblasts have H1 receptors coupled to the formation of inositol phosphates and mobilization of intracellular Ca2+. We suggest that this H1 receptor also mediates a block of the cell cycle and that histamine may play a physiological role in the regulation of fibroblast proliferation.