Growth and differentiation of stage 24 limb mesenchyme cells in a serum-free chemically defined medium

A serum-free defined medium which supports the differentiation of chick limb mesenchymal cells has been developed. In this medium, stage 24 embryonic limb mesenchymal cells which are plated at high density (5 x 10(6) cells/35-mm culture dish) differentiate into chondrocytes. Morphologically, these cultures appear only slightly different from those in which the cells are maintained in serum-containing medium. DNA levels and proline incorporation in cultures grown in defined medium are indistinguishable from control cultures. The rate of radiolabeled sulfate incorporation, a monitor of the rate of proteoglycan synthesis, in Day 8 high-density cultures maintained in defined medium is approximately 70-80% of control values. Additionally, growth and differentiation of intermediate-density (2 x 10(6) cells/35-mm culture dish) and low-density (1 x 10(6) cells/35-mm dish) cultures are also supported by this defined medium. The availability of this medium allows exploration of bioactive factors which affect or modulate mesenchymal cell differentiation and subsequent development.     

Expression of platelet-derived growth factor-beta receptors on human fibroblasts. Regulation by recombinant platelet-derived growth factor-BB, IL-1, and tumor necrosis factor-alpha.

Stimulation of human fibroblasts by platelet-derived growth factor (PDGF)-BB leads to a down-regulation of PDGF beta-receptors and a concomitant appearance of intracellular granular accumulations of receptors, as determined by stainings with the mAb PDGFR-B2. The granules contained both the ligand and PDGF beta-receptors, as revealed by double-immunofluorescence staining, and were formed in response to PDGF-BB but not in response to other cytokines tested. The formation of intracellular PDGF beta-receptor granules was dependent on PDGF-BB concentration and time of stimulation. The granular PDGF beta-receptor staining on cells treated with PDGF-BB for 1 h at 37 degrees C was used to investigate the effects of macrophage-derived cytokines on PDGF beta-receptor expression. The number of PDGF beta-receptor granules was found to be reduced in fibroblasts grown for 48 h in the presence of PDGF-BB, TNF-alpha, or IL-1; PDGF-AA under the same conditions had no effect. The reduction observed was paralleled by a decrease in cell surface expression of PDGF beta-receptors, measured as binding of 125I-PDGF-BB and of the PDGFR-B2 antibody. Furthermore, both TNF-alpha and IL-1 decreased the detergent-extractable pool of PDGF-beta receptors in the fibroblasts, as revealed by immunoblotting of detergent cell extracts. Finally, the decrease in PDGF beta-receptors after culturing of the cells in the presence of TNF-alpha and IL-1 was accompanied by a decreased incorporation of [3H]thymidine in response to PDGF-BB stimulation. In conclusion, our data suggest that certain macrophage-derived cytokines can modulate the expression of PDGF beta-receptors by cultured fibroblasts, which may contribute in part to their reduced responsiveness to PDGF.     

Site-selective dephosphorylation of the platelet-derived growth factor beta-receptor by the receptor-like protein-tyrosine phosphatase DEP-1

Ligand stimulation of PDGF beta-receptors leads to autophosphorylation of the regulatory tyrosine 857 and of tyrosine residues that in their phosphorylated form serve as docking sites for Src homology 2 domain-containing proteins. Regulation of the PDGF beta-receptor by protein-tyrosine phosphatases is poorly understood. We have investigated PDGF beta-receptor dephosphorylation by receptor-like protein-tyrosine phosphatase DEP-1 using a cell line with inducible DEP-1 expression and by characterizing in vitro dephosphorylation of the PDGF beta-receptor and of receptor-derived phosphopeptides by DEP-1. After DEP-1 induction PDGF beta-receptor.DEP-1 complexes and reduced receptor tyrosine phosphorylation were observed. Phosphopeptide analysis of the PDGF beta-receptors from DEP-1-expressing cells and of the receptors dephosphorylated in vitro by DEP-1 demonstrated that dephosphorylation of autophosphorylation sites of the receptor differed and revealed that the regulatory Tyr(P)(857) was not a preferred site for DEP-1 dephosphorylation. When dephosphorylation of synthetic receptor-derived peptides was analyzed, the selectivity was reproduced, indicating that amino acid sequence surrounding the phosphorylation sites is the major determinant of selectivity. This notion is supported by the observation that the poorly dephosphorylated Tyr(P)(562) and Tyr(P)(857), in contrast to other analyzed phosphorylation sites, are surrounded by basic amino acid residues at positions -4 and +3 relative to the tyrosine residue. Our study demonstrates that DEP-1 dephosphorylation of the PDGF beta-receptor is site-selective and may lead to modulation, rather than general attenuation, of signaling.     

Controlled Growth and the Maintenance of Human Pluripotent Stem Cells by Cultivation with Defined Medium on Extracellular Matrix-Coated Micropatterned Dishes

Here, we introduce a new serum-free defined medium (SPM) that supports the cultivation of human pluripotent stem cells (hPSCs) on recombinant human vitronectin-N (rhVNT-N)-coated dishes after seeding with either cell clumps or single cells. With this system, there was no need for an intervening sequential adaptation process after moving hPSCs from feeder layer-dependent conditions. We also introduce a micropatterned dish that was coated with extracellular matrix by photolithographic technology. This procedure allowed the cultivation of hPSCs on 199 individual rhVNT-N-coated small round spots (1 mm in diameter) on each 35-mm polystyrene dish (termed “patterned culture”), permitting the simultaneous formation of 199 uniform high-density small-sized colonies. This culture system supported controlled cell growth and maintenance of undifferentiated hPSCs better than dishes in which the entire surface was coated with rhVNT-N (termed “non-patterned cultures”). Non-patterned cultures produced variable, unrestricted cell proliferation with non-uniform cell growth and uneven densities in which we observed downregulated expression of some self-renewal-related markers. Comparative flow cytometric studies of the expression of pluripotency-related molecules SSEA-3 and TRA-1-60 in hPSCs from non-patterned cultures and patterned cultures supported this concept. Patterned cultures of hPSCs allowed sequential visual inspection of every hPSC colony, giving an address and number in patterned culture dishes. Several spots could be sampled for quality control tests of production batches, thereby permitting the monitoring of hPSCs in a single culture dish. Our new patterned culture system utilizing photolithography provides a robust, reproducible and controllable cell culture system and demonstrates technological advantages for the mass production of hPSCs with process quality control.     

Amino acid-rich medium (Leibovitz L-15) enhances and prolongs proliferation of primary cultured rat hepatocytes in the absence of serum.

Multiple rounds of cell division were induced in primary cultured rat hepatocytes in serum-free, modified L-15 medium supplemented with 20 mM NaHCO3 and 10 ng/ml EGF in a 5% CO2/95% air incubator. A 150% increase in cell number and DNA content was observed between day 1 and day 5. The time course of DNA synthesis of hepatocytes cultured in L-15 medium differed from that in DMEM/F12 medium in that there were four peaks of 3H-thymidine incorporation in the L-15 medium, at 60 h, 82 h, 96 h, and 120 h, but only one peak at 48 h in modified DMEM/F12 medium. Labeling studies of the hepatocytes indicated that more than 60% of the cells were stained with antibromodeoxyuridine (BrdU) antibody in the periods of 48-72 h and 72-96 h after plating at densities between 1.5 x 10(5) and 6.0 x 10(5) cells per 35-mm dish. Even at a density of 9.0 x 10(5) cells/dish, about 40% of the cell nuclei were stained with BrdU in the periods of 48-72 h and 72-96 h. In addition, about 20% of the hepatocytes in culture initiated a second round of the cell cycle between 48 and 96 h in culture. Proliferating cells, which were mononucleate with a little cytoplasm, appeared in small clusters or colonies in the culture from day 4. These proliferating cells produced albumin. The addition of essential amino acids to the DMEM/F12 medium enhanced the DNA synthesis of hepatocytes, thus indicating that the higher level of amino acids in L-15 medium may be an important factor in its enhanced ability to support the proliferation of primary cultured rat hepatocytes.     

Down-regulation of cellular platelet-derived growth factor receptors induced by an activated neu receptor tyrosine kinase.

The functional integration of growth factor signaling occurs at several levels in target cells. One of the most proximal mechanisms is receptor transmodulation, by which one activated receptor can regulate the expression of other receptors in the same cells. Well-established transregulatory loops involve platelet-derived growth factor (PDGF) down-regulation of epidermal growth factor (EGF) receptors and beta-type transforming growth factors modulation of PDGF receptors. We have studied the relationship between neu tyrosine kinase activation and the expression of the PDGF receptors in transfected NIH/3T3 cells. Expression of the neu oncogene, but not of the neu proto-oncogene, was associated with a decrease of PDGF alpha- and beta-receptors on the cell surface, as measured by [125-I]PDGF-AA and -BB binding. These results were corroborated by metabolic labeling and immunoprecipitation of the PDGF beta-receptors. PDGF alpha- and beta-receptor mRNAs were strongly decreased in the neu oncogene-transformed cells in comparison with control cells expressing the neu proto-oncogene. Down-regulation of the PDGF receptors and their mRNAs was also observed after EGF treatment of cells expressing a chimeric EGF receptor/neu receptor, where the neu tyrosine kinase is activated by EGF binding. These results show that the neu tyrosine kinase can down-modulate PDGF receptor expression, and the effect is mediated via decreased PDGF receptor mRNA levels.     

Estrogen-dependent expression of the chicken very low density apolipoprotein II gene in serum-free cultures of LMH cells

Summary The estrogen-responsive Leghorn strain M chicken hepatoma (LMH) cell line provides a model system for studying the estrogen-dependent, liver-specific expression of avian genes. Serum-free culture conditions have been established that allow expression of apolipoprotein B, very low density apolipoprotein II (apoVLDLII), serum albumin, and transferrin at levels detectable by Northern blot analysis. Regulation of apoVLDLII mRNA by estrogen occurred in an appropriate time-and dose-dependent manner in serum-free cultures of the LMH cells. The expression of apoVLDLII mRNA in serum-free culture was at least 100-fold higher than that expressed in cultures containing 10% serum. The level of estrogen receptors in LMH cells cultured with 10% serum was approximately 2000 receptors per cell, and in serum-free culture approximately 1000 receptors per cell. When these cells were transfected with estrogen receptor DNA and cultured in serum-free medium, apoVLDLII mRNA was decreased relative to that expressed in cells transfected with a control plasmid. These results indicate that when the LMH cells are cultured without serum, estrogen receptors are not the limiting factor for the expression of the apoVLDLII gene.     

Effect of TSH in human thyroid cells: evidence for both mitogenic and antimitogenic effects.

The well-known mitogenic effects of TSH observed in vivo on the thyroid are not always reproducible of human thyroid cells in vitro where conflicting results have been obtained. In order to clarify this issue, we have used primary cultures of human thyroid cells obtained from normal tissue and maintained in serum-free medium for several days. In this in vitro model we have studied the effect of TSH on growth by measuring three different parameters: [3H]-thymidine incorporation, cell counts, and DNA measurement. Monolayer cultures were plated at both low and high cell density (2 x 10(4) and 8 x 10(4) cells/25 mm well, respectively). Although at either cell density cultures were equally able to functionally respond to TSH in terms of cAMP accumulation a significant growth response to TSH was observed only in low density cultures. In high density cultures TSH had an antimitogenic effect. Moreover, TSH potentiated the mitogenic effect of insulin only in low density cultures. In contrast to TSH, FCS induced a similar proliferative response at both high and low cell density. Following TSH stimulation, cAMP content was always increased, paralleling the effect of growth in low density but not in high density cultures. The cAMP analogues dibutyryl-cAMP and 8-bromo-cAMP, as well as cholera toxin and forskolin, did not mimic the mitogenic effect of TSH but had an antiproliferative effect. In addition, these agents blunted the proliferative effect of insulin. These data suggest that in thyroid cells TSH is able to elicit both a mitogenic and an antimitogenic effect depending on the environmental conditions such as cell density.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stirred culture of peripheral and cord blood hematopoietic cells offers advantages over traditional static systems for clinically relevant applications

The ability to culture hematopoietic cells in readily characterizable and scalable stirred systems, combined with the capability to utilize serum-free medium, will aid the development of clinically attractive bioreactor systems for transplantation therapies. We thus examined the proliferation and differentiation characteristics of peripheral blood (PB) mononuclear cells (MNC), cord blood (CB) MNC, and PB CD34(+) cells in spinner flasks and (control) T-flask cultures in both serum-containing and serum-free media. Hematopoietic cultures initiated from all sources examined (PB MNC, CB MNC, and PB CD34(+) cells) grew well in spinner vessels with either serum-containing or serum-free medium. Culture proliferation in spinner flasks was dependent on both agitator design and RPM as well as on the establishment of critical inoculum densities (ID) in both serum-containing (2 x 10(5) MNC/mL) and serum-free (3 x 10(5) MNC/mL) media. Spinner flask culture of PB MNC in serum-containing medium provided superior expansion of total cells and colony-forming cells (CFC) at high ID (1.2 x 10(6) cells/mL) as compared to T-flask controls. Serum-free spinner culture was comparable, if not superior, to that observed in serum-containing medium. This is the first report of stirred culture of PB or CB MNC, as well as the first report of stirred CD34(+) cell culture. Additionally, this is the first account of serum-free stirred culture of hematopoietic cells from any source.     

PDGF alpha- and beta-receptors activate unique and common signal transduction pathways.

We have examined the signal transduction pathways of the PDGF alpha- and beta-receptors, in order to characterize the specificity of each receptor type in the signaling. Porcine aortic endothelial cell lines expressing equal levels of either PDGF alpha- or beta-receptors were established. The alpha- and beta-receptor cells responded mitogenically to stimulation with the proper PDGF isoforms. Three aspects of actin reorganization were examined after ligand stimulation: loss of stress fibres, appearance of edge ruffles and formation of circular membrane ruffles. The beta-receptor cells showed a response to ligand stimulation which included all three features. The alpha-receptor cells exhibited edge ruffles and loss of stress fibres, but circular ruffles could not be found in several independent alpha-receptor cell lines. The beta-receptor cells, but not the alpha-receptor cells, were able to migrate chemotactically towards a concentration gradient of ligand. The molecular basis for the differences in signalling were explored by comparing the pattern of increased phosphorylation of potential substrates for the alpha- and beta-receptors in [32P]orthophosphate labelled intact cells and using an in vitro kinase assay. Certain of the observed substrates were common for the two receptors, whereas others were specific for either one. We conclude that certain of the known PDGF induced cellular effects are transduced only by the beta-receptor; the presence of alpha-receptor-specific substrates suggests that there are also alpha-receptor-specific signals, which have yet to be identified.     

Application of a serum-free medium for the growth of Vero cells and the production of reovirus

Two strains of reovirus (serotype 1 Lang/TIL and serotype 3 Dearing/T3D) were propagated in Vero cells grown in stationary or agitated cultures in a serum-free medium, M-VSFM. Solid microcarriers (Cytodex-1) were used to support cell growth in agitated cultures with a normal doubling time of 25 h. Cell yields of 1 x 10(6) cells/mL were obtained from an inoculum of 2 x 10(5) cells/mL in 4 days in microcarrier cultures. The growth profile and cell yield was not significantly different from serum-supplemented cultures. The virus titer increased by 3-4 orders of magnitude over a culture period of 150 h. The maximum virus titer in stationary cultures reached >1 x 10(9) pfu/mL for both strains of reovirus in M-VSFM. M-VSFM also supported high viral yields in microcarrier cultures. Both the specific productivity and final viral yield was higher in M-VSFM than serum-supplemented cultures. The high viral productivity suggests that this is a suitable system for the production of reovirus as an oncolytic agent for human therapeutic use.     

Aggregate cultures of foetal rat liver cells: development and maintenance of liver gene expression

Rotation-mediated aggregate cultures of foetal rat liver cells were prepared and grown in a chemically defined medium. Their capacity for cellular organisation and maturation was studied over a culture period of 3 wk by using both morphologic and biochemical criteria. It was found that within each aggregate, distinct liver cell types were present and attained their normal, differentiated phenotype. Parenchymal cells formed small acini with a central lumen. Within the first 2 wk in culture, albumin and ferritin mRNA levels were maintained, while the alpha-fetoprotein mRNA levels decreased, and tyrosine aminotransferase (TAT) gene expression increased. No significant response to glucocorticoids was observed in early cultures, whereas after 3 wk a marked increase in TAT mRNA levels was elicited by dexamethasone and glucagon (additive stimulatory effects). The results show that foetal rat liver cells cultured in a chemically defined medium are able to rearrange themselves into histotypic structures, and display a developmental pattern of gene expression comparable to that of perinatal rat liver in vivo. This culture system offers therefore a useful model to study the development and function of liver cells.     

Ligand stimulation reduces platelet-derived growth factor beta-receptor susceptibility to tyrosine dephosphorylation

Ligand binding to the platelet-derived growth factor (PDGF) beta-receptor leads to increased receptor tyrosine phosphorylation as a consequence of dimerization-induced activation of the intrinsic receptor tyrosine kinase activity. In this study we asked whether ligand-stimulated PDGF beta-receptor tyrosine phosphorylation, to some extent, also involved reduced susceptibility to tyrosine dephosphorylation. To investigate this possibility we compared the sensitivity of ligand-stimulated and non-stimulated forms of tyrosine-phosphorylated PDGF beta-receptors to dephosphorylation using various preparations containing protein-tyrosine phosphatase activity. Ligand-stimulated or unstimulated tyrosine-phosphorylated receptors were obtained after incubation of cells with pervanadate only or pervanadate, together with PDGF-BB, respectively. Dephosphorylation of receptors immobilized on wheat germ agglutinin-Sepharose, as well as of receptors in intact cell membranes, was investigated under conditions when rephosphorylation did not occur. As compared with unstimulated receptors the ligand-stimulated PDGF beta-receptors showed about 10-fold reduced sensitivity to dephosphorylation by cell membranes, a recombinant form of the catalytic domain of density-enhanced phosphatase-1, or recombinant protein-tyrosine phosphatase 1B. We conclude that ligand-stimulated forms of the PDGF beta-receptor display a reduced susceptibility to dephosphorylation. Our findings suggest a novel mechanism whereby ligand stimulation of PDGF beta-receptor, and possibly other tyrosine kinase receptors, leads to a net increase in receptor tyrosine phosphorylation.     

Participation of beta-adrenergic receptors on macrophages in modulation of LPS-induced cytokine release

For several years it is known that beta-adrenergic receptor agonists have anti-inflammatory effects. However, little is known about the role of beta-adrenergic receptors on macrophages in the modulation of cytokine production by beta-agonists during inflammation. In this study, the presence of beta-receptors on PMA-differentiated U937 human macrophages, and the participation of these receptors in the modulation of LPS-mediated cytokine production by beta-agonists was investigated. Total beta-receptor expression on undifferentiated (monocyte) and PMA-differentiated U937 cells was established using receptor binding studies on membrane fractions with a radio ligand. The expression of beta-receptors proved to be significantly lower on monocytes than on macrophages, additionally a predominant expression of beta 2-receptors was found. Production of the cytokines TNF-alpha, IL-6, and IL-10 by LPS-stimulated differentiated U937 cells was measured in time. Peak concentrations for TNF-alpha, IL-6 and IL-10 occurred at 3, 12 and 9 hrs, respectively. When differentiated U937 cells were incubated with both LPS and the beta-agonist clenbuterol the production of TNF-alpha and IL-6 was significantly reduced. However the production of IL-10 was increased. To study the mechanism of modulation of cytokine production in more detail, U937 macrophages were incubated with LPS/clenbuterol in combination with selective beta 1- and beta 2-antagonists. These results indicated that the beta 2- and not the beta 1-receptor is involved in the anti-inflammatory activity of clenbuterol.     

Serum-free collagen sandwich cultures of adult rat hepatocytes maintain liver-like properties long term: A valuable model for in vitro toxicity and drug-drug interaction studies

Cultures of primary hepatocytes from various species, including human, are used in several applications during pre-clinical drug development. Their use is however limited by cell survival and conservation of liver-specific functions in vitro. The differentiation status of hepatocytes in culture strongly depends on medium formulation and the extracellular matrix environment. We incubated primary rat hepatocytes for 10 days on collagen monolayer and in collagen sandwich cultures with or without serum. Restoration of polygonal cell shape and formation of functional bile canaliculi-like structures was stable only in serum-free sandwich cultures. Variations in general cell viability, as judged by the cellular ATP content, LDH release or apoptosis, were less pronounced between alternative cultures. The intracellular glutathione content was preserved close to in vivo levels especially in serum-free sandwich cultures. Basal activities of cytochrome P450 enzymes (P450) varied strongly between cultures. There was a minor effect on CYP1A but CYP2B activity was only detectable in the serum-free sandwich culture after 3 days and beyond. CYP2C activity was slightly elevated in both sandwich cultures, whereas CYP3A showed increased levels in both serum-free cultures. Inducibility of these P450s was fully maintained over time in serum-free collagen sandwich only. Gene expression was largely constant over time in serum-free sandwich cultures that was closest to liver. This liver-like property was supported by protein profiling results. Taken together, the serum-free collagen sandwich culture of primary rat hepatocytes maintained liver-like features over 10 days and is therefore a suitable model for long-term toxicity and drug-drug interaction studies.     

Transferrin is an autocrine growth factor secreted by reuber H-35 cells in serum-free culture

Summary We have previously reported that Reuber H-35 rat hepatoma cells secrete an autocrine growth-stimulating activity in serum-free culture. To characterize this activity, conditioned serum-free medium from dense H-35 donor cultures was collected in the absence and presence of [³⁵S]methionine. A 1∶4 dilution of conditioned medium into fresh serum-free medium resulted in an increase in mean H-35 cell numbers per assay dish from 1.59±0.12×10⁵ to 3.35±0.34×10⁵ after 44 h of incubation. Control, unconditioned medium, resulted in significantly (P<0.05) less growth (2.14±0.41×10⁵ cells per dish). Trypsin digestion eliminated the growth-promoting effect of conditioned medium but had no effect on unconditioned medium. Dialysis did not diminish the growth-promoting activity of conditioned medium. The immunoprecipitate of [³⁵S]methionine-containing conditioned medium with antisera against rat serum transferrin contained a dominant radioactive doublet of molecular weight equal to purified rat serum transferrin. A rat transferrin radioimmunoassay was devised and used to quantitate that 29.1±1.2 ng of transferrin was secreted per 10⁶ cells per hour in serum-free culture. Addition of antitransferrin antibody resulted in a significant (P<0.025) decrease in H-35 cell growth after 48 h. Thus, a portion of the autocrine growth-promoting activity secreted by H-35 cells into serum-free culture is due to transferrin. This work was funded by a feasibility grant from the American Diabetes Association, as well as by grants CA 24604-09 and CA 16463-14 from The National Institutes of Health, Bethesda, MD.     

Neuronal Activation Regulates the Expression of Opioid Receptors: Possible Role of Glial-Derived Factors and Voltage-Dependent Ion Channels

The previous observation that a continuous chemical depolarization of aggregating rat brain cells with KCl alters the expression of opioid receptors was examined in more detail. In contrast to its significant and converse effect on forebrain and hindbrain cells cultured in serum-containing medium, KCl had only a small and transient effect in serum-free cultures of both types. The basal receptor density in serum-free cultures was similar to the receptor density in KCl-treated serum-containing cultures, but medium conditioned by glial cells restored partially the effect of KCl in serum-free cultures. The effect of KCl in serum-containing forebrain cultures was enhanced by the voltage-dependent calcium channel blocker verapamil, and magnesium and cadmium had a similar, though smaller, effect. The sodium channel activator veratridine had a profound and dose-dependent inhibitory effect on the expression of the receptors in forebrain and hindbrain cultures, and tetrodotoxin blocked the veratridine effect. Information about the selectivity of the effect of neuronal activation on the various opioid receptor subtypes was obtained with the neuroblastoma X glioma hybrid M8 cells that possess only delta type opioid receptors. A Scatchard analysis of [3H]etorphine binding to these cells has shown that depolarization increased the Bmax, but had little, if any, effect on the affinity (KD) of the ligand to the receptors. The significance of depolarization and voltage-dependent sodium and calcium channels on the expression of different opioid receptor subtypes is discussed.     

Influence of in vitro plating density on rat granulosa cell responsiveness to follicle-stimulating hormone

The effects of cell plating density on granulosa cell sensitivity to follicle-stimulating hormone (FSH) were investigated, using a serum-free culture of cells obtained from immature, estrogen-treated rats. The cells were incubated at densities of 0.25 to 5 X 10(5) cells/dish with increasing concentrations of FSH for 2 days, and medium estrogen and progestin accumulation were measured by radioimmunoassay. Per-cell estrogen and progestin production rose with increasing FSH concentration and cell density up to 2 X 10(5) cells/dish. At a higher density (5 X 10(5)/dish), per-cell estrogen production fell; progestin production remained constant, although the major progestin produced was no longer progesterone, but rather its metabolite, 20 alpha-hydroxy-progesterone. The effects of changing cell density could not be accounted for by medium steroids or cytotoxic substances. It is concluded that in vitro plating density can markedly affect granulosa cell sensitivity to FSH. In vivo, changing intrafollicular cell densities may thus affect the ability of the whole cell complement to respond to gonadotropin.     

Loss of T-cell protein tyrosine phosphatase induces recycling of the platelet-derived growth factor (PDGF) beta-receptor but not the PDGF alpha-receptor

We have previously shown that the T-cell protein tyrosine phosphatase (TC-PTP) dephosphorylates the platelet-derived growth factor (PDGF) beta-receptor. Here, we show that the increased PDGF beta-receptor phosphorylation in TC-PTP knockout (ko) mouse embryonic fibroblasts (MEFs) occurs primarily on the cell surface. The increased phosphorylation is accompanied by a TC-PTP-dependent, monensin-sensitive delay in clearance of cell surface PDGF beta-receptors and delayed receptor degradation, suggesting PDGF beta-receptor recycling. Recycled receptors could also be directly detected on the cell surface of TC-PTP ko MEFs. The effect of TC-PTP depletion was specific for the PDGF beta-receptor, because PDGF alpha-receptor homodimers were cleared from the cell surface at the same rate in TC-PTP ko MEFs as in wild-type MEFs. Interestingly, PDGF alphabeta-receptor heterodimers were recycling. Analysis by confocal microscopy revealed that, in TC-PTP ko MEFs, activated PDGF beta-receptors colocalized with Rab4a, a marker for rapid recycling. In accordance with this, transient expression of a dominant-negative Rab4a construct increased the rate of clearance of cell surface receptors on TC-PTP ko MEFs. Thus, loss of TC-PTP specifically redirects the PDGF beta-receptor toward rapid recycling, which is the first evidence of differential trafficking of PDGF receptor family members.     

Differential expression of platelet-derived growth factor receptors in human malignant glioma cell lines.

Glioma cells in culture express platelet-derived growth factor (PDGF) A- and B-chains and secrete PDGF-like activity that is mainly PDGF-AA. In this work, we show that the PDGF alpha- and beta-receptors are independently expressed in human malignant glioma cells. We also define three different receptor phenotypes that are related to the morphology of glioma cells: cells with only alpha-receptors, only beta-receptors, or with both types of receptors. By the help of Northern blot analyses, 125I-PDGF-binding experiments, and immunoprecipitations the receptors are shown to be structurally normal PDGF receptors, except for minor variations in size that probably are due to differences in glycosylation. PDGF-BB induces DNA synthesis in cells of all three receptor phenotypes, whereas PDGF-AA or PDGF-AB has this effect only on cells with alpha- or with alpha- and beta-receptors. 125I-PDGF-AB binds with high affinity and down-regulates beta-receptors only in cells where alpha-receptors are present in addition to beta-receptors. Thus, the different functional capacities of PDGF isoforms on glioma cells fit with their known receptor-binding specificities and are compatible with the hypothesis that the isoforms act by inducing dimeric receptor complexes. When data on PDGF A- and B-chains, as well as alpha- and beta-receptor expression are compiled and the pattern of receptor binding specificity is taken into account, the majority of glioma cell lines are found to have a phenotype that makes autocrine stimulation possible.