Deletion of the PDGFR-beta gene affects key fibroblast functions important for wound healing

This study provides new perspectives of the unique aspects of platelet-derived growth factor beta-receptor (PDGFR-beta) signaling and biological responses through the establishment of a mutant mouse strain in which two loxP sequences were inserted into the introns of PDGFR-beta genome sequences. Isolation of skin fibroblasts from the mutant mice and Cre recombinase transfection in vitro induced PDGFR-beta gene deletion (PDGFR-betaDelta/Delta). The resultant depletion of the PDGFR-beta protein significantly attenuated platelet-derived growth factor (PDGF)-BB-induced cell migration, proliferation, and protection from H2O2-induced apoptosis of the cultured PDGFR-betaDelta/Delta dermal fibroblasts. PDGF-AA and fetal bovine serum were mitogenic and anti-apoptotic but were unable to induce the migration in PDGFR-beta Delta/Delta fibroblasts. Concerning the PDGF signaling, PDGF-BB-induced phosphorylation of Akt, ERK1/2, and JNK, but not p38, decreased in PDGFR-betaDelta/Delta fibroblasts, but PDGF-AA-induced signaling was not altered. Overexpression of the phospholipid phosphatases, SHIP2 and/or PTEN, inhibited PDGF-BB-induced phosphorylation of Akt and ERK1/2 in PDGFR-betaDelta/Delta fibroblasts but did not affect that of JNK and p38. These results indicate that disruption of distinct PDGFR-beta signaling pathways in PDGFR-betaDelta/Delta dermal fibroblasts impaired their proliferation and survival, but completely inhibits migratory response, and that PDGF-BB-induced phosphorylation of Akt and ERK1/2 possibly mediated by PDGFR-alpha is regulated, at least in part, by the lipid phosphatases SHIP2 and/or PTEN. Thus, the PDGFR-beta function on dermal fibroblasts appears to be critical in PDGF-BB action for skin wound healing and is clearly distinctive from that of PDGFR-alpha in the ligand-induced biological responses and the underlying properties of cellular signaling.     

Both platelet-derived growth factor receptor (PDGFR)-alpha and PDGFR-beta promote murine fibroblast cell migration

Cell motility plays a critical role for many physiological and pathological processes including wound healing, fibrosis, angiogenesis, and tumor metastasis. Platelet-derived growth factor (PDGF) is among the most potent stimuli for mesenchymal cell migration. The PDGF B-chain homodimer PDGF BB activates both alpha- and beta-receptor subunits (alpha-PDGFR and beta-PDGFR), and promotes cell migration in many cell types including fibroblasts and smooth muscle cells. PDGF-A chain homodimer PDGF AA activates alpha-PDGFR only, and its role for cell migration is still debatable. PDGF BB, but not PDGF AA, induces smooth muscle cell migration. Interestingly, alpha-PDGFR was shown to antagonize beta-PDGFR-induced smooth muscle cell migration. In the present study, we investigated the role of alpha-PDGFR and beta-PDGFR in PDGF-mediated cell migration of murine fibroblasts (NIH 3T3). Unlike smooth muscle cells, both PDGF AA and PDGF BB promoted NIH 3T3 cell migration. The effect of PDGF BB activation of beta-PDGFR alone for cell migration was examined using previously established NIH 3T3 clones in which alpha-PDGFR signaling is inhibited by a dominant-negative alpha-PDGFR, or an antisense construct of alpha-PDGFR. PDGF BB activation of beta-PDGFR alone was sufficient to induce cell migration, but the efficiency was significantly lower compared to PDGF activation of both receptors. These results showed that both alpha- and beta-PDGFRs promote fibroblast cell migration and their effects are additive. Taken together, we propose that cell-type specific alpha-PDGFR signaling is critical for regulation of mesenchymal cell migration in response to PDGF isoform, whereas beta-PDGFR mainly promotes cell migration.     

Lysyl oxidase oxidizes cell membrane proteins and enhances the chemotactic response of vascular smooth muscle cells

Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied cell types. Here we demonstrate that inhibition of LOX activity by beta-aminopropionitrile (BAPN) in cultured rat aortic smooth muscle cells (SMCs) reduced the chemotactic response and sensitivity of these cells toward LOX and toward PDGF-BB. The chemotactic activity of PDGF-BB was significantly enhanced in the presence of a non-chemotactic concentration of LOX. We considered the possibility that extracellular LOX may oxidize cell surface proteins, including the PDGF receptor-beta (PDGFR-beta), to affect PDGF-BB-induced chemotaxis. Plasma membranes purified from control SMC contained oxidized PDGFR-beta. The oxidation of this receptor and other membrane proteins was largely prevented in cells preincubated with BAPN. Addition of purified LOX to these cells restored the profile of oxidized proteins toward that of control cells. The high affinity and capacity for the binding of PDGF-BB by cells containing oxidized PDGFR-beta was diminished by approximately 2-fold when compared with cells in which oxidation by LOX was prevented by BAPN. Phosphorylated members of the PDGFR-beta-dependent signal transduction pathway, including PDGFR-beta, SHP2, AKT1, and ERK1/ERK2 (p44/42 MAPK), turned over faster in BAPN-treated than in control SMCs. LOX knock-out mouse embryonic fibroblasts mirrored the effect obtained with SMCs treated with BAPN. These novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR-beta.     

Lycopene inhibits PDGF-BB-induced signaling and migration in human dermal fibroblasts through interaction with PDGF-BB

In melanoma development and progression, platelet-derived growth factor (PDGF) has been suggested to modulate the microenvironment, especially stromal fibroblasts, to the benefit of melanoma growth, invasion, and metastasis. Lycopene, a natural carotenoid that is abundant in tomato, has been shown to inhibit proliferation of several types of cancer cells. However, little attention has been paid to skin fibroblasts and melanoma cells. In the present study, we determined the effects of lycopene on stromal fibroblasts and their interactions with melanoma cells. We found that lycopene inhibited PDGF-BB-induced human Hs68 skin fibroblast migration on gelatin and collagen. Further analysis showed that lycopene inhibited PDGF-BB-induced signaling in human Hs68 and primary cultured skin fibroblasts. PDGF-BB-induced phosphorylation of PDGF receptor beta (PDGFR-beta), extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK) was attenuated by lycopene in a concentration-dependent manner, whereas the total expression of each protein was not affected. Interestingly, dot binding assay revealed that lycopene could directly bind to human PDGF-BB in PBS and human plasma, indicating that lycopene can bind to PDGF-BB in both in vitro and in vivo conditions. In functional studies, lycopene inhibited melanoma-induced fibroblast migration in a noncontact coculture system and attenuated signaling in fibroblasts simulated by melanoma-derived conditioned medium. Our results provide the first evidence showing that lycopene is an effective inhibitor of migration of stromal fibroblasts and this effect may contribute to its antitumor activity.     

PDGF-D is a specific, protease-activated ligand for the PDGF beta-receptor

The term 'platelet-derived growth factor' (PDGF) refers to a family of disulphide-bonded dimeric isoforms that are important for growth, survival and function in several types of connective tissue cell. So far, three different PDGF chains have been identified - the classical PDGF-A and PDGF-B and the recently identified PDGF-C. PDGF isoforms (PDGF-AA, AB, BB and CC) exert their cellular effects by differential binding to two receptor tyrosine kinases. The PDGF alpha-receptor (PDGFR-alpha) binds to all three PDGF chains, whereas the beta-receptor (PDGFR-beta) binds only to PDGF-B. Gene-targeting studies using mice have shown that the genes for PDGF-A and PDGF-B, as well as the two PDGFR genes, are essential for normal development. Furthermore, overexpression of PDGFs is linked to different pathological conditions, including malignancies, atherosclerosis and fibroproliferative diseases. Here we have identify and characterize a fourth member of the PDGF family, PDGF-D. PDGF-D has a two-domain structure similar to PDGF-C and is secreted as a disulphide-linked homodimer, PDGF-DD. Upon limited proteolysis, PDGF-DD is activated and becomes a specific agonistic ligand for PDGFR-beta. PDGF-DD is the first known PDGFR-beta-specific ligand, and its unique receptor specificity indicates that it may be important for development and pathophysiology in several organs.     

Expression patterns of PDGF-A, -B, -C and -D and the PDGF-receptors alpha and beta in activated rat hepatic stellate cells (HSC)

The platelet-derived growth factor (PDGF) family, which regulates many physiological and pathophysiological processes has recently been enlarged by two new members, the isoforms PDGF-C and -D. Little is known about the expression levels of these new members in hepatic fibrosis. We therefore investigated by quantitative real time PCR (Taqman) the mRNA expression profiles of all four PDGF isoforms in transdifferentiating primary cultured hepatic stellate cells (HSC), an in vitro model system of hepatic fibrogenesis, either with or without stimulation of the cells with PDGF-BB or TGF-beta1. All four isoforms were expressed in HSC transdifferentiating to myofibroblast-like cells (MFB) albeit with different profiles: while PDGF-A mRNA exhibited minor fluctuations only, PDGF-B was rapidly down-regulated. In contrast, both PDGF-C and -D mRNA were strongly induced: PDGF-C up to 5 fold from day 2 to day 8 and PDGF-D up to 8 fold from day 2 to day 5 of culture. Presence of PDGF-DD in activated HSC was confirmed at the protein level by immunocytochemistry. Stimulation of HSC and MFB with PDGF-BB led to down-regulation of the new isoforms, whereas TGF-beta1 upregulated PDGF-A only. We further show that PDGF receptor-beta (PDGFR-beta) mRNA was rapidly upregulated within the first day of culture and was constantly expressed from day 2 on while the expression profile of PDGFR-alpha mRNA was very similar to that of PDGF-A during transdifferentiation. Given the dramatic changes in PDGF-C and -D expression, which may compensate for down-regulation of PDGF-B, we hypothesize that the new PDGF isoforms may fulfil specific functions in hepatic fibrogenesis.     

PDGF-BB-mediated activation of p42(MAPK) is independent of PDGF beta-receptor tyrosine phosphorylation

Herein, we investigated the activity of mitogen-activated protein kinase (MAPK), a key component of downstream signaling events, which is activated subsequent to platelet-derived growth factor (PDGF)-BB stimulation. Specifically, p42(MAPK) activity peaked 60 min after addition of PDGF-BB, declined thereafter, and was determined not to be a direct or necessary component of glycosaminoglycan (GAG) synthesis. PDGF-BB also activated MAPK kinase 2 (MAPKK2) but had no effect on MAPKK1 and Raf-1 activity. Chemical inhibition of Janus kinase, phosphatidylinositol 3-kinase, Src kinase, or tyrosine phosphorylation inhibition of the PDGF beta-receptor (PDGFR-beta) did not abrogate PDGF-BB-induced p42(MAPK) activation or its threonine or tyrosine phosphorylation. A dominant negative cytoplasmic receptor for hyaluronan-mediated motility variant 4 (RHAMMv4), a regulator of MAPKK-MAPK interaction and activation, did not inhibit PDGF-BB-induced p42(MAPK) activation nor did a construct expressing PDGFR-beta with cytoplasmic tyrosines mutated to phenylalanine. However, overexpression of a dominant negative PDGFR-beta lacking the cytoplasmic signaling domain abrogated p42(MAPK) activity. These results suggest that PDGF-BB-mediated activation of p42(MAPK) requires the PDGFR-beta but is independent of its tyrosine phosphorylation.     

Angiotensin II and noradrenaline increase PDGF-BB receptors and potentiate PDGF-BB stimulated DNA synthesis in vascular smooth muscle

The effects of angiotensin II and noradrenaline were examined on PDGF-BB and PDGF-AB induced mitogenesis in primary cultures of rat aortic smooth muscle. Incubation of the smooth muscle with either angiotensin II or noradrenaline potentiated the submaximal but not maximal mitogenic effects of PDGF-BB but not PDGF-AB. These effects on PDGF-BB stimulated mitogenesis correlated with an increase in receptor number specific for this homodimer when the smooth muscle was incubated with either angiotensin II or noradrenaline. Mitogenic concentrations of PDGF-AB did not interact with this PDGF receptor subtype. These results indicate that the mitogenic effects of PDGF-AB and -BB are elicited via different PDGF receptor subtypes. Angiotensin II and noradrenaline potentiate the mitogenic effects of PDGF-BB by increasing the steady state concentrations of membrane receptors for this homodimer.     

Inhibition of platelet-derived growth factor-mediated signal transduction by transforming ras. Suppression of receptor autophosphorylation.

The function of ras protein and its relationship to growth-factor mediated signal transduction remain unclear. The demonstration that the expression of transforming ras (v-ras) can block the stimulation of growth-related gene expression and cell division mediated by the platelet-derived growth factor (PDGF) may provide a model for the functional interation of ras with growth factor receptors. In the current studies, we have demonstrated that this blockade by v-ras of PDGF-BB signal transduction occurs very early in signal transduction, at the level of PDGF receptor autophosphorylation. Although the expression of PDGF receptor as detected by Western blot with anti-PDGF receptor antibody was not diminished in v-ras-transformed murine Balb/c 3T3 fibroblasts, the autophosphorylation of PDGF receptor in response to ligand (recombinant PDGF-BB homodimer) stimulation was profoundly suppressed. This same phenomenon of v-ras-mediated PDGF receptor autophosphorylation inhibition was also demonstrated in normal rat kidney fibroblasts. Further, factor(s) present in v-ras-expressing fibroblasts found in the membrane fractions of these cells can dominantly inhibit the autophosphorylation of the PDGF receptor obtained from normal fibroblasts. These findings suggest a role for ras in one of the earliest steps of the signal transduction pathway.     

Lack of pericytes leads to endothelial hyperplasia and abnormal vascular morphogenesis

The association of pericytes (PCs) to newly formed blood vessels has been suggested to regulate endothelial cell (EC) proliferation, survival, migration, differentiation, and vascular branching. Here, we addressed these issues using PDGF-B-- and PDGF receptor-beta (PDGFR-beta)--deficient mice as in vivo models of brain angiogenesis in the absence of PCs. Quantitative morphological analysis showed that these mutants have normal microvessel density, length, and number of branch points. However, absence of PCs correlates with endothelial hyperplasia, increased capillary diameter, abnormal EC shape and ultrastructure, changed cellular distribution of certain junctional proteins, and morphological signs of increased transendothelial permeability. Brain endothelial hyperplasia was observed already at embryonic day (E) 11.5 and persisted throughout development. From E 13.5, vascular endothelial growth factor-A (VEGF-A) and other genes responsive to metabolic stress became upregulated, suggesting that the abnormal microvessel architecture has systemic metabolic consequences. VEGF-A upregulation correlated temporally with the occurrence of vascular abnormalities in the placenta and dilation of the heart. Thus, although PC deficiency appears to have direct effects on EC number before E 13.5, the subsequent increased VEGF-A levels may further abrogate microvessel architecture, promote vascular permeability, and contribute to formation of the edematous phenotype observed in late gestation PDGF-B and PDGFR-beta knock out embryos.     

Low density lipoprotein receptor-related protein 1 (LRP1) controls endocytosis and c-CBL-mediated ubiquitination of the platelet-derived growth factor receptor beta (PDGFR beta)

The low density lipoprotein receptor-related protein 1 (LRP1) has been implicated in intracellular signaling functions as well as in lipid metabolism. Recent in vivo and in vitro studies suggest that LRP1 is a physiological modulator of the platelet-derived growth factor (PDGF) signaling pathway. Here we show that in mouse fibroblasts LRP1 modulates PDGF-BB signaling by controlling endocytosis and ligand-induced down-regulation of the PDGF receptor beta (PDGFRbeta). In LRP1-deficient fibroblasts, basal PDGFRbeta tyrosine kinase activity was derepressed, and PDGF-BB-induced endocytosis and degradation of PDGFRbeta were accelerated as compared with control cells. This was accompanied by rapid uptake of receptor-bound PDGF-BB into the cells and by attenuated ERK activation in response to PDGF-BB stimulation. Pulse-chase analysis indicated that the steady-state turnover rate of PDGFRbeta was also accelerated in LRP-deficient fibroblasts. The rapid degradation of PDGFRbeta in the LRP1-deficient fibroblasts was prevented by MG132 and chloroquine. Furthermore, the association of PDGFRbeta with c-Cbl, a ubiquitin E3-ligase, as well as the ligand-induced ubiquitination of PDGFRbeta were increased in LRP1-deficient fibroblasts. We show that LRP1 can directly interact with c-Cbl, suggesting a Sprouty-like role for LRP1 in regulating the access of the PDGFRbeta to the ubiquitination machinery. Thus, LRP1 modulates PDGF signaling by controlling ubiquitination and endocytosis of the PDGFRbeta.     

Relative binding and biochemical effects of heterodimeric and homodimeric isoforms of platelet-derived growth factor in osteoblast-enriched cultures from fetal rat bone

Platelet-derived growth factor (PDGF) exists as a homodimer or a heterodimer comprising either PDGF-A or PDGF-B subunits, and each isoform occurs in various tissues, including bone. Although the stimulatory effects of PDGF-BB have been studied in cultures of bone cells and intact bone fragments, the influence of other isoforms that may arise locally or systematically in vivo, has not been reported. Therefore recombinant human PDGF-BB, PDGF-AB, and PDGF-AA were evaluated in osteoblast-enriched cultures from fetal rat bone. Within 24 hours these factors produced a graded response in bone cell DNA and protein synthesis, with half-maximal effects at approximately 0.6, 2.1, and 4.8 nM PDGF-BB, PDGF-AB, and PDGF-AA, respectively. Increases in collagen and noncollagen protein synthesis were abrogated when DNA synthesis was blocked with hydroxyurea. Furthermore, each factor reduced alkaline phosphatase activity, PDGF-BB being the most inhibitory. Binding studies with 125I-PDGF-BB or 125I-PDGF-AA and each unlabeled PDGF isoform produced discrete ligand binding and displacement patterns: 125I-PDGF-BB binding was preferentially displaced by PDGF-BB (Ki approximately 0.7 nM), less by PDGF-AB (Ki approximately 2.3 nM) and poorly by PDGF-AA. In contrast, 125I-PDGF-AA binding was measurably reduced by PDGF-AA (Ki approximately 4.0 nM), but was more effectively displaced by PDGF-BB or PDGF-AB (each with Ki approximately 0.7 nM). These studies indicate that each PDGF isoform produces biochemical effects proportional to binding site occupancy and suggest that receptors that favor PDGF-B subunit binding preferentially mediate these results in osteoblast-enriched bone cell cultures.     

PDGF-BB induces vascular smooth muscle cell expression of high molecular weight FGF-2, which accumulates in the nucleus

Basic fibroblast growth factor (FGF-2) and platelet-derived growth factor (PDGF) are implicated in vascular remodeling secondary to injury. Both growth factors control vascular endothelial and smooth muscle cell proliferation, migration, and survival through overlapping intracellular signaling pathways. In vascular smooth muscle cells PDGF-BB induces FGF-2 expression. However, the effect of PDGF on the different forms of FGF-2 has not been elucidated. Here, we report that treatment of vascular aortic smooth muscle cells with PDGF-BB rapidly induces expression of 20.5 and 21 kDa, high molecular weight (HMW) FGF-2 that accumulates in the nucleus and nucleolus. Conversely, PDGF treatment has little or no effect on 18 kDa, low-molecular weight FGF-2 expression. PDGF-BB-induced upregulation of HMW FGF-2 expression is controlled by sustained activation of extracellular signal-regulated kinase (ERK)-1/2 and is abolished by actinomycin D. These data describe a novel interaction between PDGF-BB and FGF-2, and indicate that the nuclear forms of FGF-2 may mediate the effect of PDGF activity on vascular smooth muscle cells.     

Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse.

Development of a vascular system involves the assembly of two principal cell types - endothelial cells and vascular smooth muscle cells/pericytes (vSMC/PC) - into many different types of blood vessels. Most, if not all, vessels begin as endothelial tubes that subsequently acquire a vSMC/PC coating. We have previously shown that PDGF-B is critically involved in the recruitment of pericytes to brain capillaries and to the kidney glomerular capillary tuft. Here, we used desmin and alpha-smooth muscle actin (ASMA) as markers to analyze vSMC/PC development in PDGF-B-/- and PDGFR-beta-/- embryos. Both mutants showed a site-specific reduction of desmin-positive pericytes and ASMA-positive vSMC. We found that endothelial expression of PDGF-B was restricted to immature capillary endothelial cells and to the endothelium of growing arteries. BrdU labeling showed that PDGFR-beta-positive vSMC/PC progenitors normally proliferate at sites of endothelial PDGF-B expression. In PDGF-B-/- embryos, limb arterial vSMC showed a reduced BrdU-labeling index. This suggests a role of PDGF-B in vSMC/PC cell proliferation during vascular growth. Two modes of vSMC recruitment to newly formed vessels have previously been suggested: (1) de novo formation of vSMC by induction of undifferentiated perivascular mesenchymal cells, and (2) co-migration of vSMC from a preexisting pool of vSMC. Our data support both modes of vSMC/PC development and lead to a model in which PDGFR-beta-positive vSMC/PC progenitors initially form around certain vessels by PDGF-B-independent induction. Subsequent angiogenic sprouting and vessel enlargement involves PDGF-B-dependent vSMC/PC progenitor co-migration and proliferation, and/or PDGF-B-independent new induction of vSMC/PC, depending on tissue context.     

PDGF-induced Akt phosphorylation does not activate NF-kappa B in human vascular smooth muscle cells and fibroblasts

A recent report suggested that platelet-derived growth factor (PDGF) activates nuclear factor-kappa B (NF-kappa B) by phosphorylation of the protein kinase Akt [Romashkova and Makarov, Nature 401 (1999) 86-90]. The present study investigates the role of Akt in the activation of NF-kappa B by tumor necrosis factor-alpha (TNF alpha, 10 ng/ml) and PDGF-BB (20 ng/ml) in human vascular smooth muscle cells (SMC), skin and foreskin fibroblasts. TNF alpha stimulated serine phosphorylation and degradation of the inhibitory protein I kappa B alpha and strongly induced nuclear NF-kappa B translocation and binding activity. PDGF did not induce serine phosphorylation or degradation of I kappa B alpha and did not enhance binding activity of NF-kappa B. In contrast, stimulation with PDGF resulted in a marked phosphorylation of Akt, but no Akt phosphorylation occurred after stimulation with TNF alpha. These data suggest that Akt phosphorylation is not involved in NF-kappa B activation in human SMC and fibroblasts.     

Actions of platelet-derived growth factor isoforms in mesangial cells

Platelet-derived growth factor (PDGF) occurs as homodimers or heterodimers of related polypeptide chains PDGF-BB, -AA, and -AB. There are two receptors that bind PDGF, termed alpha and beta. The beta receptor recognizes PDGF B chain and is dimerized in response to PDGF BB. The alpha receptor recognizes PDGF B as well as A chains and can be dimerized by the three dimeric forms of PDGF AA, AB, and BB. To characterize PDGF receptor signaling mechanisms and biologic activities in human mesangial cells (MC), we explored the effects of the three PDGF isoforms on DNA synthesis, phospholipase C activation, and PDGF protooncogene induction. PDGF-BB homodimer and AB heterodimer induced a marked increase in DNA synthesis, activation of phsopholipase C, and autoinduction of PDGF A and B chain mRNAs, whereas PDGF-AA homodimer was without effect. The lack of response to PDGF AA could be accounted for by down regulation of the PDGF-alpha receptor since preincubation of MC with suramin restored PDGF AA-induced DNA synthesis. Ligand binding studies demonstrate specific binding of labeled PDGF BB and AB and to a lower extent PDGF AA isoforms to mesangial cells. These results are consistent with predominant expression of PDGF beta receptor in MC, which is linked to phospholipase-C activation. The potent biologic effects of PDGF-AB heterodimer in cells that express very few alpha receptors and do not respond to PDGF AA are somewhat inconsistent with the currently accepted model of PDGF receptor interaction and suggest the presence of additional mechanisms for PDGF isoform binding and activation. © 1994 Wiley-Liss, Inc.