A receptor-like inositol lipid phosphatase is required for the maturation of developing cochlear hair bundles.

A screen for protein tyrosine phosphatases (PTPs) expressed in the chick inner ear yielded a high proportion of clones encoding an avian ortholog of protein tyrosine phosphatase receptor Q (Ptprq), a receptor-like PTP. Ptprq was first identified as a transcript upregulated in rat kidney in response to glomerular nephritis and has recently been shown to be active against inositol phospholipids. An antibody to the intracellular domain of Ptprq, anti-Ptprq, stains hair bundles in mice and chicks. In the chick ear, the distribution of Ptprq is almost identical to that of the 275 kDa hair-cell antigen (HCA), a component of hair-bundle shaft connectors recognized by a monoclonal antibody (mAb) that stains inner-ear hair bundles and kidney glomeruli. Furthermore, anti-Ptprq immunoblots a 275 kDa polypeptide immunoprecipitated by the anti-HCA mAb from the avian inner ear, indicating that the HCA and Ptprq are likely to be the same molecule. In two transgenic mouse strains with different mutations in Ptprq, anti-Ptprq immunoreactivity cannot be detected in the ear. Shaft connectors are absent from mutant vestibular hair bundles, but the stereocilia forming the hair bundle are not splayed, indicating that shaft connectors are not necessary to hold the stereocilia together; however, the mice show rapid postnatal deterioration in cochlear hair-bundle structure, associated with smaller than normal transducer currents with otherwise normal adaptation properties, a progressive loss of basal-coil cochlear hair cells, and deafness. These results reveal that Ptprq is required for formation of the shaft connectors of the hair bundle, the normal maturation of cochlear hair bundles, and the long-term survival of high-frequency auditory hair cells.

PTPRQ is a novel phosphatidylinositol phosphatase that can be expressed as a cytoplasmic protein or as a subcellularly localized receptor-like protein.

PTPRQ (rPTP-GMC1) is a member of the type III receptor-like protein tyrosine phosphatase family. PTPRQ has very low activity against phosphotyrosine but is active against phosphatidylinositol phosphates that are involved in regulation of survival, proliferation, and subcellular architecture. Here, we report that PTPRQ can be expressed as a cytosolic or a receptor-like protein and that the form, subcellular localization, and cell types in which it is expressed are regulated by alternative promoter use and by alternative splicing. The first promoter drives expression of transcripts encoding a transmembrane protein in human podocytes and lung. PTPRQ protein is localized to the basal membrane of human podocytes, beginning when podocyte progenitors can first be identified in the embryonic kidney. A second promoter drives expression of a transcript that can encode a cytoplasmic protein containing the catalytic site. This is the major PTPRQ transcript in rat mesangial cells and human testis and is upregulated in mesangial cells in a rat model of mesangial proliferative glomerulonephritis. Differential regulation of expression of the transmembrane vs cytosolic forms, in different cell types during development or response to injury, may be a mechanism through which PTPRQ, with its activities against membrane phospholipids and against phosphotyrosine, can target specific substrates under different conditions.

Protein tyrosine phosphatase RQ is a phosphatidylinositol phosphatase that can regulate cell survival and proliferation.

Protein tyrosine phosphatase RQ (PTPRQ) was initially identified as a protein tyrosine phosphatase (PTPase)-like protein that is upregulated in a model of renal injury. Here we present evidence that, like PTEN, the biologically important enzymatic activity of PTPRQ is as a phosphatidylinositol phosphatase (PIPase). The PIPase specificity of PTPRQ is broader than that of PTEN and depends on different amino acid residues in the catalytic domain. In vitro, the recombinant catalytic domain of PTPRQ has low PTPase activity against tyrosine-phosphorylated peptide and protein substrates but can dephosphorylate a broad range of phosphatidylinositol phosphates, including phosphatidylinositol 3,4,5-trisphosphate and most phosphatidylinositol monophosphates and diphosphates. Phosphate can be hydrolyzed from the D3 and D5 positions in the inositol ring. PTPRQ does not have either of the basic amino acids in the catalytic domain that are important for the PIPase activity of PTEN or the sequence motifs that are characteristic of type II phosphatidylinositol 5-phosphatases. Instead, the PIPase activity depends on the WPE sequence present in the catalytic cleft of PTPRQ, and in the "inactive" D2 domains of many dual-domain PTPases, in place of the WPD motif present in standard active PTPases. Overexpression of PTPRQ in cultured cells inhibits proliferation and induces apoptosis. An E2171D mutation that retains or increases PTPase activity but eliminates PIPase activity, eliminates the inhibitory effects on proliferation and apoptosis. These results indicate that PTPRQ represents a subtype of the PTPases whose biological activities result from its PIPase activity rather than its PTPase activity.

Platelet-derived growth factor B-chain of hematopoietic origin is not necessary for granulation tissue formation and its absence enhances vascularization.

The hypothesis that wound repair is augmented by delivery of platelet-derived growth factor (PDGF) from platelets and macrophages is an attractive extrapolation from the known activities of PDGF in cell culture and in vivo. To test this hypothesis in mice, we prepared hematopoietic chimeras, in which the hematopoietic system of a normal adult mouse was replaced by the hematopoietic system of a PDGF B-chain -/- or +/+ donor. We initiated local granulation tissue formation either by implanting small surgical sponges to elicit a foreign body granulation tissue response, or by ligating the left common carotid to form an organized thrombus. We found that the absence of hematopoietic PDGF B-chain did not decrease the extent of granulation tissue or vascular lesion formation, and that the vascularization of both lesions increased by approximately 100%. We conclude that PDGF B-chain from cells of hematopoietic origin, including platelets and macrophages, is not important for granulation tissue formation, and that it reduces vascularization of granulation issue, probably through disabling of the short-range chemotactic gradients of PDGF that are important for recruiting pericytes/smooth muscle cells to the endothelium of new vessels.

ADAM15 overexpression in NIH3T3 cells enhances cell-cell interactions.

ADAM15 is a member of the family of metalloprotease-disintegrins that have been shown to interact with integrins in an RGD- and non-RGD-dependent manner. In the present study, we examined the effects of ADAM15 overexpression on cell-matrix and cell-cell interactions in NIH3T3 cells. Tetracycline-regulated ADAM15 overexpression in NIH3T3 cells leads to an inhibition of migration on a fibronectin-coated filter in a Boyden chamber assay and in a scratch wound model. The effects of ADAM15 overexpression on cell migration are not due to changes in matrix attachment or to the lack of extracellular signal-regulated kinase signaling response to PDGF or fibronectin. However, a decrease in monolayer permeability with ADAM15 overexpression and altered cell morphology suggest a possible increase in cell-cell interaction. Analysis of adhesion of NIH3T3 cells to a polyclonal population of cells retrovirally transduced to overexpress ADAM15 demonstrates a 45% increase in cell adhesion, compared with enhanced green fluorescent protein-expressing control cells. In addition, we demonstrate localization of HA-epitope-tagged ADAM15 to cell-cell contacts in an epithelial cell line that forms extensive cell-cell contact structures. Thus, overexpression of ADAM15 in NIH3T3 cells appears to enhance cell-cell interactions, as suggested by decreased cell migration, altered cell morphology at the wound edge, decreased monolayer permeability, and increased cell adhesion to monolayers of cells expressing ADAM15 by retroviral transduction.

Control of shape and size of vascular smooth muscle cells in vitro by plasma lithography.

The ability to control the shape and size of cells is an important enabling technique for investigating influences of geometrical variables on cell physiology. Herein we present a micropatterning technique ("plasma lithography") that uses photolithography and plasma thin-film polymerization for the fabrication of cell culture substrates with a cell-adhesive pattern on a cell-repellent (non-fouling) background. The micron-level pattern was designed to isolate individual vascular smooth muscle cells (SMC) on areas with a projected area of between 25 and 3600 microm(2) in order to later study their response to cytokine stimulation in dependence of the cell size and shape as an indication for the phenotypic state of the cells. Polyethylene terephthalate substrates were first coated with a non-fouling plasma polymer of tetraglyme (tetraethylene glycol dimethyl ether). In an organic lift-off process, we then fashioned square- and rectangular-shaped islands of a thin fluorocarbon plasma polymer film of approximately 12-nm thickness. Electron spectroscopy for chemical analysis and secondary ion mass spectroscopy were used to optimize the deposition conditions and characterize the resulting polymers. Secondary ion mass spectroscopy imaging was used to visualize the spatial distribution of the polymer components of the micropatterned surfaces. Rat vascular SMC were seeded onto the patterned substrates in serum-free medium to show that the substrates display the desired properties, and that cell shape can indeed be controlled. For long-term maintenance of these cells, the medium was augmented with 10% calf serum after 24 h in culture, and the medium was exchanged every 3 days. After 2 weeks, the cells were still confined to the areas of the adhesive pattern, and when one or more cells spanned more than one island, they did not attach to the intervening tetraethylene glycol dimethyl ether (tetraglyme) background. Spreading-restricted cells formed a well-ordered actin skeleton, which was most dense along the perimeter of the cells. The shape of the nucleus was also influenced by the pattern geometry. These properties make the patterned substrates suitable for investigating if the phenotypic reversion of SMC can be influenced by controlling the shape and size of SMC in vitro.

Apoptosis of smooth muscle cells is not silent: Fas/FADD initiates a program of inflammatory gene expression.

Unlike necrosis, apoptosis is classically considered to be "silent," (i.e., self-contained and non-inflammatory). In this review, we describe the system that we developed to regulate apoptosis of smooth muscle cells (SMC) in vivo. We have used this system to demonstrate that SMC apoptosis initiated by FADD or by Fas ligation includes a specific program of expression of pro-inflammatory genes. We discuss how this conclusion can be reconciled with reports that Fas plays an anti-inflammatory role in vascular lesions.

Basis of hematopoietic defects in platelet-derived growth factor (PDGF)-B and PDGF beta-receptor null mice.

Platelet-derived growth factor (PDGF)-B and PDGF beta-receptor (PDGFR beta) deficiency in mice is embryonic lethal and results in cardiovascular, renal, placental, and hematologic disorders. The hematologic disorders are described, and a correlation with hepatic hypocellularity is demonstrated. To explore possible causes, the colony-forming activity of fetal liver cells in vitro was assessed, and hematopoietic chimeras were demonstrated by the transplantation of mutant fetal liver cells into lethally irradiated recipients. It was found that mutant colony formation is equivalent to that of wild-type controls. Hematopoietic chimeras reconstituted with PDGF-B(-/-), PDGFR beta(-/-), or wild-type fetal liver cells show complete engraftment (greater than 98%) with donor granulocytes, monocytes, B cells, and T cells and display none of the cardiovascular or hematologic abnormalities seen in mutants. In mouse embryos, PDGF-B is expressed by vascular endothelial cells and megakaryocytes. After birth, expression is seen in macrophages and neurons. This study demonstrates that hematopoietic PDGF-B or PDGFR beta expression is not required for hematopoiesis or integrity of the cardiovascular system. It is argued that metabolic stress arising from mutant defects in the placenta, heart, or blood vessels may lead to impaired liver growth and decreased production of blood cells. The chimera models in this study will serve as valuable tools to test the role of PDGF in inflammatory and immune responses. (Blood. 2001;97:1990-1998)

Endothelial cells of hematopoietic origin make a significant contribution to adult blood vessel formation.

Granulation tissue formation is an example of new tissue development in an adult. Its rich vascular network has been thought to derive via angiogenic sprouting and extension of preexisting vessels from the surrounding tissue. The possibility that circulating cells of hematopoietic origin can differentiate into vascular endothelial cells (ECs) in areas of vascular remodeling has recently gained credibility. However, no quantitative data have placed the magnitude of this contribution into a physiological perspective. We have used hematopoietic chimeras to determine that 0.2% to 1.4% of ECs in vessels in control tissues derived from hematopoietic progenitors during the 4 months after irradiation and hematopoietic recovery. By contrast, 8.3% to 11.2% of ECs in vessels that developed in sponge-induced granulation tissue during 1 month derived from circulating hematopoietic progenitors. This recruitment of circulating progenitors to newly forming vessels would be difficult to observe in standard histological studies, but it is large enough to be encouraging for attempts to manipulate this contribution for therapeutic gain.

Fas/FADD-mediated activation of a specific program of inflammatory gene expression in vascular smooth muscle cells.

Apoptosis of smooth muscle cells is a common feature of vascular lesions but its pathophysiological significance is not known. We demonstrate that signals initiated by regulated Fas-associated death domain protein overexpression in rat vascular smooth muscle cells in the carotid artery induce expression of monocyte-chemoattractant protein-1 and interleukin-8, and cause massive immigration of macrophages in vivo. These chemokines, and a specific set of other pro-inflammatory genes, are also upregulated in human vascular smooth muscle cells during Fas-induced apoptosis, in part through a process that requires interleukin-1alpha activation. Induction of a pro-inflammatory program by apoptotic vascular smooth muscle cells may thus contribute to the pathogenesis of vascular disease.

Protein-tyrosine phosphatases in the vessel wall: differential expression after acute arterial injury.

Many protein-tyrosine phosphatases (PTPases) have now been identified, but little is known about PTPase expression and regulation in vascular tissue and in vascular disease. Polymerase chain reaction (PCR) amplification and cDNA fingerprinting of PTPase catalytic domains, combined with random sequencing of PCR product libraries, identified 18 (8 receptor-like and 10 cytosolic) PTPases in the rat carotid artery and revealed differential expression of 5 of these PTPases during neointima formation after balloon catheter injury. In situ hybridization was used to localize mRNA expression in vessel cross sections for the 5 differentially expressed PTPases. This revealed that for 3 PTPases (SHP1, CD45, and PTPbeta), differential transcript abundance was due to appearance/loss of the cell types by which they were expressed (leukocytes for SHP1 and CD45, endothelial cells for PTPbeta). However, mRNA expression of 2 PTPases (PTPL1 and PTP1B) was specifically upregulated by proliferating and migrating smooth muscle cells (SMCs) in characteristic temporal and regional patterns in response to vessel damage. Quantitative PCR analysis showed that PTP1B and PTPL1 were induced approximately 30-fold and approximately 60-fold, respectively, by 2 weeks after injury in the damaged vessels compared with the uninjured vessels. PTP1B was rapidly upregulated in the media after vessel injury and remained highly expressed in the developing neointima. By contrast, PTPL1 expression did not increase dramatically until the SMCs had migrated into the intima. The differential expression of PTP1B and PTPL1 by SMCs after injury suggests roles for these PTPases in the regulation of vessel wall remodeling.

Matrix metalloproteinase-9 overexpression enhances vascular smooth muscle cell migration and alters remodeling in the injured rat carotid artery.

Matrix metalloproteinase-9 (MMP-9) has been implicated in the pathogenesis of atherosclerosis as well as intimal hyperplasia after vascular injury. We used Fischer rat smooth muscle cells (SMCs) overexpressing MMP-9 to determine the role of MMP-9 in migration and proliferation as well as in vessel remodeling after balloon denudation. Fischer rat SMCs were stably transfected with a cDNA for rat MMP-9 under the control of a tetracycline-regulatable promoter. In this system, MMP-9 was overexpressed in the absence, but not in the presence, of tetracycline. In vitro SMC migration was determined using a collagen invasion assay as well as a Boyden chamber assay. In vivo migration was determined by measuring the invasion into the medial and intimal layers of transduced SMCs seeded on the outside of the artery. Transduced SMCs were also seeded on the luminal surface, and the effect of local MMP-9 overexpression on vascular structure was measured morphometrically at intervals up to 28 days. MMP-9 overexpression enhanced SMC migration in both the collagen invasion assay and Boyden chamber in vitro, increased SMC migration into an arterial matrix in vivo, and altered vessel remodeling by increasing the vessel circumference, thinning the vessel wall and decreasing intimal matrix content. These results demonstrate that MMP-9 enhances vascular SMC migration in vitro and in vivo and alters postinjury vascular remodeling.

The Tel-PDGFRbeta fusion gene produces a chronic myeloproliferative syndrome in transgenic mice.

Chronic myelomonocytic leukemia (CMML) is a pre-leukemic syndrome that displays both myelodysplastic and myeloproliferative features. The t(5;12) chromosomal translocation, present in a subset of CMML patients with myeloproliferation fuses the amino terminal portion of the ets family member, Tel, with the transmembrane and tyrosine kinase domains of platelet-derived growth factor receptor beta (PDGFRbeta) gene. To investigate the role of this fusion protein in the pathogenesis of CMML, we expressed the Tel-PDGFRbeta fusion cDNA in hematopoietic cells of transgenic mice under the control of the human CD11a promoter. Transgenic founders and their offspring express the transgene specifically in hematopoietic tissues and develop a myeloproliferative syndrome characterized by: overproduction of mature neutrophils and megakaryocytes in the bone marrow; splenomegaly with effacement of splenic architecture by extramedullary hematopoiesis; an abnormal population of leukocytes co-expressing lymphoid and myeloid markers; and increased numbers of colonies in in vitro bone marrow CFU assays. All mice expressing the transgene exhibited at least one of these features of dysregulated myelopoiesis, and 20% progressed to a myeloid or lymphoid malignancy. This murine model of CMML parallels a myeloproliferative syndrome in humans and implicates the Tel-PDGFRbeta fusion protein in its pathogenesis.

Chimera analysis reveals that fibroblasts and endothelial cells require platelet-derived growth factor receptorbeta expression for participation in reactive connective tissue formation in adults but not during development.

The hypothesis that platelet-derived growth factor (PDGF) plays an important role in repair of connective tissue has been difficult to test experimentally, in part because the disruption of any of the PDGF ligand and receptor genes is embryonic lethal. We have developed a method that circumvents the embryonic lethality of the PDGF receptor (R)beta-/- genotype and minimizes the tendency of compensatory processes to mask the phenotype of gene disruption by comparing the behavior of wild-type and PDGFRbeta-/- cells within individual chimeric mice. This quantitative chimera analysis method has revealed that during development PDGFRbeta expression is important for all muscle lineages but not for fibroblast or endothelial lineages. Here we report that fibroblasts and endothelial cells, but not leukocytes, are dependent on PDGFRbeta expression during the formation of new connective tissue in and around sponges implanted under the skin. Even the 50% reduction in PDGFRbeta gene dosage in PDGFRbeta+/- cells reduces fibroblast and endothelial cell participation by 85%. These results demonstrate that the PDGFRbeta/PDGF B-chain system plays an important direct role in driving both fibroblast and endothelial cell participation in connective tissue repair, that cell behavior can be regulated by relatively small changes in PDGFRbeta expression, and that the functions served by PDGF in wound healing are different from the roles served during development.

Proliferating and migrating mesangial cells responding to injury express a novel receptor protein-tyrosine phosphatase in experimental mesangial proliferative glomerulonephritis.

The mesangial cell provides structural support to the kidney glomerulus. A polymerase chain reaction-based cDNA display approach identified a novel protein-tyrosine phosphatase, rPTP-GMC1, whose transcript expression is transiently and dramatically up-regulated during the period of mesangial cell migration and proliferation that follows mesangial cell injury in the anti-Thy 1 model of mesangial proliferative glomerulonephritis in the rat. In situ hybridization analysis confirmed that rPTP-GMC1 mRNA is up-regulated specifically by mesangial cells responding to the injury and is not detectable in other cells in the kidney or in many normal tissues. In cell culture, rPTP-GMC1 is expressed by mesangial cells but not by glomerular endothelial or epithelial cells (podocytes). The longest transcript (7.5 kilobases) encodes a receptor-like protein-tyrosine phosphatase consisting of a single catalytic domain, a transmembrane segment, and 18 fibronectin type III-like repeats in the extracellular segment. A splice variant predicts a truncated molecule missing the catalytic domain. rPTP-GMC1 maps to human chromosome 12q15 and to the distal end of mouse chromosome 10. The predicted structure of rPTP-GMC1 and its pattern of expression in vivo and in culture suggest that it plays a role in regulating the adhesion and migration of mesangial cells in response to injury.

Expression of platelet-derived growth factor and its receptors in the developing and adult mouse kidney.

BACKGROUND: Experimental analysis of gene function is increasingly being accomplished using mouse models. Glomerular malformations occur in mice in which the platelet-derived growth factor (PDGF) B-chain gene or the PDGF receptor beta-subunit gene have been deleted. To understand potential PDGF signaling pathways in the kidney, we determined the expression pattern of PDGF ligand and receptor genes in mouse kidney during development and in the mature adult kidney. METHODS: We used in situ hybridization to map the expression of transcripts encoding the PDGF ligands (A-chain and B-chain) and PDGF receptors (PDGFRalpha and PDGFRbeta) in the developing and mature kidney of the mouse. RESULTS: PDGF A-chain transcripts are expressed by epithelial cells (especially in what appear to be the loop of Henle) and possibly in vascular smooth muscle cells. Its receptor, PDGFRalpha, is expressed by interstitial cells. PDGF B-chain transcripts are most highly expressed by vascular endothelial cells of developing and adult kidney and minimally by visceral epithelia of immature glomeruli. PDGFRbeta transcripts are expressed by fetal blastemal cells, interstitial cells, mesangial cells, and vascular smooth muscle cells and by adult mesangial and interstitial cells. PDGFRalpha and PDGFRbeta expression is especially prominent in lipid-laden interstitial cells in the adult kidney. CONCLUSIONS: These patterns of expression are similar, but not identical, to those observed in rat and human and suggest that paracrine interactions mediated by the PDGF/PDGF receptor system may coordinate the development of the tubular, vascular, and interstitial components during kidney development and disease.

Chimaeric analysis reveals role of Pdgf receptors in all muscle lineages.

Blood vessels originate as simple endothelial cell tubes. It has been proposed that platelet-derived growth factor B polypeptide (Pdgfb) secreted by these endothelial cells drives the formation of the surrounding muscular wall by recruiting nearby mesenchymal cells. However, targetted inactivation of the Pdgfb gene or the Pdgf receptor beta (Pdgfrb) gene, by homologous recombination, does not prevent the development of apparently normal large arteries and connective tissue. We have used an in vivo competition assay in which we prepared chimaeric blastocysts, composed of a mixture of wild-type (Pdgfrb[+/+]) and Pdgfrb(+/-) or wild-type and Pdgfrb(-/-) cells, and quantified the relative success of cells of the two component genotypes in competing for representation in different cell lineages as the chimaeric embryos developed. This study revealed that the participation of Pdgfrb(-/-) cells in all muscle lineages (smooth, cardiac, skeletal and pericyte) was reduced by eightfold compared with Pdgfrb(+/+) cells, and that participation of Pdgfrb(+/-) cells was reduced by twofold (eightfold for pericytes). Pdgfrb inactivation did not affect cell contribution to non-muscle mesodermal lineages, including fibroblasts and endothelial cells. Chimaera competition is therefore a sensitive, quantitative method for determining developmental roles of specific genes, even when those roles are not apparent from analysis of purebred mutants; most likely because they are masked by homeostatic mechanisms.

Extraglomerular origin of the mesangial cell after injury. A new role of the juxtaglomerular apparatus.

We investigated the origin of the glomerular mesangial cell, a smooth muscle-like cell that provides structural support in the glomerulus. Injection of anti-Thy 1 antibody that binds the Thy 1 antigen on rat mesangial cells eliminated (> 95%) the mesangial population at 20-28 h, while Thy 1-positive cells in the juxtaglomerular apparatus (JGA) were sequestered from the circulation and survived. Single pulse labeling with [3H]thymidine at 36 h labeled Thy 1-positive cells in the JGA and hilus. Serial biopsies demonstrated the progressive migration (5-15 micron/d) and proliferation of these mesangial reserve cells until the entire glomerulus was repopulated. The regenerating mesangial population expressed contractile and migratory proteins preferentially at the leading edge of the migratory front. Single as well as multiple pulse labeling with [3H]thymidine confirmed that the entire mesangial cell repopulation originated from only a few mesangial reserve cells. These reserve cells resided in the extraglomerular mesangium in the JGA and were not renin-secreting cells, macrophages, smooth muscle cells, or endothelial cells. These studies document mesangial cell migration in the anti-Thy 1 model of mesangial proliferative glomerulonephritis and provide evidence for a new role for the juxtaglomerular apparatus in the maintenance of the mesangial cell population.

Localization of PDGF alpha-receptor in the developing and mature human kidney.

Using in situ hybridization and immunocytochemistry we describe the renal localization of the PDGF alpha-receptor. PDGF alpha-receptor mRNA was uniformly present in human metanephric kidney in interstitial cells and vascular arcades that course through the blastema. PDGF alpha-receptor mRNA was present in some mesangial structures in early glomeruli, but was largely lost as glomeruli matured. It was present in adventitial fibroblasts, but usually not in vascular smooth muscle cells or endothelial cells of the fetal vasculature. This pattern persisted in adult kidneys, with extensive expression of mRNA by interstitial cells and only occasional expression by mesangial cells. All in situ hybridization findings were corroborated by immunocytochemistry. Double immunolabeling confirmed the rare expression of the PDGF alpha-receptor protein by vascular smooth muscle cells and the absence of its expression by endothelial cells. Given that both PDGF A- and B-chain can promote smooth muscle cell and fibroblast migration and proliferation and that both signal through the PDGF alpha-receptor, these data suggest that PDGF alpha-receptor may play important roles in the early vasculogenesis of the fetal kidney as well as in the pathogenesis of renal interstitial fibrosis.

Regulation of platelet-derived growth factor receptor expression by cell context overrides regulation by cytokines.

Immunocytochemical data has indicated that platelet-derived growth factor receptor beta-subunit (PDGFR beta) expression by connective tissue cells is up-regulated in many disease states. To investigate potential causes of this up-regulation, we have evaluated conditions that regulate PDGF receptor transcript levels in cultured diploid human fibroblast model systems. We found combinations of soluble mediators and cell "context," which can regulate receptor transcripts (and receptor protein) over a 50-fold range, with cell context factors being far more potent regulators than soluble mediators. For cells grown under standard monolayer conditions on plastic, levels of both PDGFR beta and PDGFR alpha increase 10-fold as culture density increases. Cells grown in suspension or in three-dimensional gels express 10- to 20-fold higher transcript levels than cells plated on plastic at comparable density and serum concentration. The soluble mediators tested, including 14 cytokines and conditioned medium from activated lymphocytes, have only modest effects on transcript levels. Lymph decreases PDGFR beta transcript expression 4-fold, suggesting that a component of interstitial fluid contributes to maintenance of the low basal level of expression in normal tissues. The mitogenic responsiveness of cells cultured at different densities parallels the level of PDGFR beta expression. Blocking anti-PDGF receptor antibodies decrease receptor availability and mitogenic responsiveness in parallel. In both cases, the striking overlap between the PDGF-BB binding and mitogenesis dose-response curves suggests that the level of PDGF receptor expression can limit responsiveness to PDGF. Overall, these results suggest that the up-regulation of PDGF receptor expression seen under pathological conditions may be due to disruption of the cell's normal environment/context/cell shape/cell attachment and that this could serve to ensure that a proliferative response to PDGF would occur only under conditions in which there had been significant tissue damage.