Galectin-3 promotes the adipogenic differentiation of PDGFRα+ cells and ectopic fat formation in regenerating muscle.

Worldwide prevalence of obesity is associated with the increase of lifestyle-related diseases. The accumulation of intermuscular adipose tissue (IMAT) is considered a major problem whereby obesity leads to sarcopenia and metabolic disorders and thus is a promising target for treating these pathological conditions. However, whereas obesity-associated IMAT is suggested to originate from PDGFRα+ mesenchymal progenitors, the processes underlying this adipogenesis remain largely unexplored. Here, we comprehensively investigated intra- and extracellular changes associated with these processes using single-cell RNA sequencing and mass spectrometry. Our single-cell RNA sequencing analysis identified a small PDGFRα+ cell population in obese mice directed strongly toward adipogenesis. Proteomic analysis showed that the appearance of this cell population is accompanied by an increase in galectin-3 in interstitial environments, which was found to activate adipogenic PPARγ signals in PDGFRα+ cells. Moreover, IMAT formation during muscle regeneration was significantly suppressed in galectin-3 knockout mice. Our findings, together with these multi-omics datasets, could unravel microenvironmental networks during muscle regeneration highlighting possible therapeutic targets against IMAT formation in obesity.

Platelet-derived growth factor receptor-α is essential for cardiac fibroblast survival.

Platelet-derived growth factor receptor α (PDGFRα), a receptor tyrosine kinase required for cardiac fibroblast development, is uniquely expressed by fibroblasts in the adult heart. Despite the consensus that PDGFRα is expressed in adult cardiac fibroblasts, we know little about its function when these cells are at rest. Here, we demonstrate that loss of PDGFRα in cardiac fibroblasts resulted in a rapid reduction of resident fibroblasts. Furthermore, we observe that phosphatidylinositol 3-kinase signaling was required for PDGFRα-dependent fibroblast maintenance. Interestingly, this reduced number of fibroblasts was maintained long-term, suggesting that there is no homeostatic mechanism to monitor fibroblast numbers and restore hearts to wild-type levels. Although we did not observe any systolic functional changes in hearts with depleted fibroblasts, the basement membrane and microvasculature of these hearts were perturbed. Through in vitro analyses, we showed that PDGFRα signaling inhibition resulted in an increase in fibroblast cell death, and PDGFRα stimulation led to increased levels of the cell survival factor activating transcription factor 3. Our data reveal a unique role for PDGFRα signaling in fibroblast maintenance and illustrate that a 50% loss in cardiac fibroblasts does not result in lethality.NEW & NOTEWORTHY Platelet-derived growth factor receptor α (PDGFRα) is required in developing cardiac fibroblasts, but a functional role in adult, quiescent fibroblasts has not been identified. Here, we demonstrate that PDGFRα signaling is essential for cardiac fibroblast maintenance and that there are no homeostatic mechanisms to regulate fibroblast numbers in the heart. PDGFR signaling is generally considered mitogenic in fibroblasts, but these data suggest that this receptor may direct different cellular processes depending on the cell's maturation and activation status.

Selective deletion of hepatocyte platelet-derived growth factor receptor α and development of liver fibrosis in mice.

BACKGROUND: Platelet-derived growth factor receptor α (PDGFRα) expression is increased in activated hepatic stellate cells (HSCs) in cirrhotic liver, while normal hepatocytes express PDGFRα at a negligible level. However, cancerous hepatocytes may show upregulation of PDGFRα, and hepatocellular carcinoma is preceded by chronic liver injury. The role of PDGFRα in non-cancerous hepatocytes and liver fibrosis is unclear. We hypothesized that upon liver injury, PDGFRα in insulted hepatocytes contributes to liver fibrosis by facilitating intercellular crosstalk between hepatocytes and HSCs. METHODS: Hepatocytes were isolated from normal and thioacetamide (TAA)-induced cirrhotic livers for assessment of PDGFRα expression. Conditional knock-out (KO) C57BL/6 mice, in which PDGFRα was selectively deleted in hepatocytes, were generated. Liver fibrosis was induced by injecting TAA for 8 weeks. Hep3B cells were transfected with a small interfering RNA (siRNA) (PDGFRα or control) and co-cultured with LX2 cells. RESULTS: PDGFRα expression was increased in hepatocytes from fibrotic livers compared to normal livers. Conditional PDGFRα KO mice had attenuated TAA-induced liver fibrosis with decreased HSC activation and proliferation. Immunoblot analyses revealed decreased expression of phospho-p44/42 MAPK in TAA-treated KO mice; these mice also showed almost complete suppression of the upregulation of mouse double minute 2. Although KO mice exhibited increased expression of transforming growth factor (TGF)-ß and Smad2/3, this was compensated for by increased expression of inhibitory Smad7. LX2 cells co-cultured with PDGFRα siRNA-infected Hep3B cells showed decreased PDGFRα, α smooth muscle actin, collagen α1(I), TGFß, and Smad2/3 expression. LX2/PDGFRα-deleted hepatocyte co-culture medium showed decreased PDGF-BB and PDGF-CC levels. CONCLUSIONS: Deletion of PDGFRα in hepatocytes attenuated the upregulation of PDGFRα in HSCs after TAA treatment, resulting in decreased liver fibrosis and HSC activation. This suggests that in the event of chronic liver injury, PDGFRα in hepatocytes plays an important role in liver fibrosis by affecting PDGFRα expression in HSCs.

Deficiency of platelet-derived growth factor receptor-α-positive cells in Hirschsprung's disease colon.

AIM: To investigate whether the expression of platelet-derived growth factor receptor-α-positive (PDGFRα(+))-cells is altered in Hirschsprung's disease (HD). METHODS: HD tissue specimens (n = 10) were collected at the time of pull-through surgery, while colonic control samples were obtained at the time of colostomy closure in patients with imperforate anus (n = 10). Immunolabelling of PDGFRα(+)-cells was visualized using confocal microscopy to assess the distribution of these cells, while Western blot analysis was undertaken to quantify PDGFRα protein expression. RESULTS: Confocal microscopy revealed PDGFRα(+)-cells within the mucosa, myenteric plexus and smooth muscle in normal controls, with a marked reduction in PDGFRα(+)-cells in the HD specimens. Western blotting revealed high levels of PDGFRα protein expression in normal controls, while there was a striking decrease in PDGFRα protein expression in the HD colon. CONCLUSION: These findings suggest that the altered distribution of PDGFRα(+)-cells in both the aganglionic and ganglionic HD bowel may contribute to the motility dysfunction in HD.

RNA interference against platelet-derived growth factor receptor α mRNA inhibits fibroblast transdifferentiation in skin lesions of patients with systemic sclerosis.

OBJECTIVE: To down-regulate expression of mRNA for the platelet-derived growth factor receptor (PDGFR)-α, block the signalling pathway of PDGF and its receptor, and study their influence on fibroblast transdifferentiation to myofibroblasts in systemic sclerosis (SSc). METHODS: Fibroblasts from skin lesions of SSc patients and health adult controls were cultured in vitro, and α-smooth muscle actin (α-SMA) expression was determined by immunocytochemistry. Both groups of fibroblasts were stimulated with PDGF-AA, transforming growth factor ß1 (TGF-ß1), and costimulated with PDGF-AA and TGF-ß1, then PDGFR-α and α-SMA mRNA and protein expression were detected with RT-PCR and WB respectively. Three pairs of siRNAs targeting different PDGFR-α mRNA sequences were synthesized for RNAi. SSc and control fibroblasts were transfected with PDGFR-α siRNA; stimulated with PDGF-AA; and assessed for PDGFR-α and α-SMA mRNA and protein expression. RESULTS: Although the fibroblasts from both groups had similar morphology, the SSc skin lesions had significantly more myofibroblasts than control skin lesions. PDGF-AA stimulation, TGF-ß1 stimulation, and costimulation significantly up-regulated PDGFR-α and α-SMA mRNA and protein expression in SSc fibroblasts compared to control (P<0.05), and costimulation had the strongest effects (P<0.05). All three pairs of siRNAs suppressed PDGFR-α mRNA and protein expression (P<0.05), but siRNA1495 had the highest gene-silencing efficiency (P<0.05). PDGFR-α siRNA attenuated the effects of PDGF-AA through up-regulating PDGFR-α and α-SMA mRNA and protein expression and inhibiting fibroblast transdifferentiation to myofibroblasts in SSc (P<0.05). CONCLUSIONS: PDGFR-α over-expression in SSc fibroblasts bound PDGF-AA more efficiently and promoted fibroblast transdifferentiation, which was enhanced by TGF-ß1. PDGFR-α siRNA down-regulated PDGFR-α expression, blocked binding to PDGF-AA, and inhibited fibroblast transdifferentiation to myofibroblasts.

Differential mechanisms of acquired resistance to insulin-like growth factor-i receptor antibody therapy or to a small-molecule inhibitor, BMS-754807, in a human rhabdomyosarcoma model.

Agents targeting insulin-like growth factor-I receptor (IGF-IR), including antibodies and small-molecule inhibitors, are currently in clinical development for the treatment of cancers including sarcoma. However, development of resistance is a common phenomenon resulting in failures of anticancer therapies. In light of this problem, we developed two resistant models from the rhabdomyosarcoma cell line Rh41: Rh41-807R, with acquired resistance to BMS-754807, a small-molecule dual-kinase inhibitor targeting IGF-IR and insulin receptor (IR), and Rh41-MAB391R, with resistance to MAB391, an IGF-IR-blocking antibody. In addition, tumor xenograft models were established from Rh41 and Rh41-807R cell lines. Gene expression and DNA copy number analyses of these models revealed shared as well as unique acquired resistance mechanisms for the two types of IGF-IR inhibitors. Each resistant model used different signaling pathways as a mechanism for proliferation. Platelet-derived growth factor receptor α (PDGFRα) was amplified, overexpressed, and constitutively activated in Rh41-807R cells and tumors. Knockdown of PDGFRα by small interfering RNA in Rh41-807R resensitized the cells to BMS-754807. Synergistic activities were observed when BMS-754807 was combined with PDGFRα inhibitors in the Rh41-807R model in vitro. In contrast, AXL expression was highly elevated in Rh41-MAB391R but downregulated in Rh41-807R. Notably, BMS-754807 was active in Rh41-MAB391R cells and able to overcome resistance to MAB391, but MAB391 was not active in Rh41-807R cells, suggesting potentially broader clinical activity of BMS-754807. This is the first study to define and compare acquired resistance mechanisms for IGF-IR-targeted therapies. It provides insights into the differential acquired resistance mechanisms for IGF-IR/IR small-molecule inhibitor versus anti-IGF-IR antibody.

Platelet-derived growth factor-alpha receptor activation is required for human cytomegalovirus infection.

Human cytomegalovirus (HCMV) is a ubiquitous human herpesvirus that can cause life-threatening disease in the fetus and the immunocompromised host. Upon attachment to the cell, the virus induces robust inflammatory, interferon- and growth-factor-like signalling. The mechanisms facilitating viral entry and gene expression are not clearly understood. Here we show that platelet-derived growth factor-alpha receptor (PDGFR-alpha) is specifically phosphorylated by both laboratory and clinical isolates of HCMV in various human cell types, resulting in activation of the phosphoinositide-3-kinase (PI(3)K) signalling pathway. Upon stimulation by HCMV, tyrosine-phosphorylated PDGFR-alpha associated with the p85 regulatory subunit of PI(3)K and induced protein kinase B (also known as Akt) phosphorylation, similar to the genuine ligand, PDGF-AA. Cells in which PDGFR-alpha was genetically deleted or functionally blocked were non-permissive to HCMV entry, viral gene expression or infectious virus production. Re-introducing human PDGFRA gene into knockout cells restored susceptibility to viral entry and essential viral gene expression. Blockade of receptor function with a humanized PDGFR-alpha blocking antibody (IMC-3G3) or targeted inhibition of its kinase activity with a small molecule (Gleevec) completely inhibited HCMV viral internalization and gene expression in human epithelial, endothelial and fibroblast cells. Viral entry in cells harbouring endogenous PDGFR-alpha was competitively inhibited by pretreatment with PDGF-AA. We further demonstrate that HCMV glycoprotein B directly interacts with PDGFR-alpha, resulting in receptor tyrosine phosphorylation, and that glycoprotein B neutralizing antibodies inhibit HCMV-induced PDGFR-alpha phosphorylation. Taken together, these data indicate that PDGFR-alpha is a critical receptor required for HCMV infection, and thus a target for novel anti-viral therapies.

PDGF and FGF2 pathways regulate distinct oligodendrocyte lineage responses in experimental demyelination with spontaneous remyelination.

Repair of myelin damage in the adult CNS requires oligodendrocyte progenitor (OP) proliferation and subsequent differentiation into remyelinating oligodendrocytes. Platelet-derived growth factor (PDGF) and fibroblast growth factor-2 (FGF2) have been predicted to act individually and/or cooperatively to generate remyelinating oligodendrocytes. Analysis of PDGF alpha receptor (PDGF alpha R) heterozygous (+/-) mice indicates that PDGF alpha R expression modulates oligodendrocyte density in non-lesioned adult CNS. Analysis of cuprizone demyelination and recovery in PDGF alpha R+/- mice, FGF2 knockout (-/-) mice, and PDGF alpha R+/- FGF2-/- mice demonstrated that: (1) OP proliferation and oligodendrocyte regeneration is impaired in PDGF alpha R heterozygotes, (2) PDGF alpha R+/- and FGF2-/- deletions do not act cooperatively to impair OP amplification, (3) oligodendrocyte differentiation is more frequent in FGF2-/- mice, and (4) FGF2 deletion in combination with the PDGF alpha R+/- genotype rescues impaired oligodendrocyte regeneration of PDGF alpha R heterozygotes. These findings demonstrate distinct roles for PDGF and FGF2 in vivo in the context of a demyelinating disease with spontaneous remyelination.

PDGF alpha-receptor signal strength controls an RTK rheostat that integrates phosphoinositol 3'-kinase and phospholipase Cgamma pathways during oligodendrocyte maturation.

Receptors with tyrosine kinase activity (RTKs) control tissue growth and development in metazoans. How they generate cell-specific responses remains essentially unknown; one model proposes that distinct RTKs activate different second-messenger pathways, whereas a second proposes that all RTKs deliver a generic "go" signal to these pathways that is uniquely interpreted by downstream, cell-specific response competence factors. We examine pathway activation and pathway-specific responses downstream of PDGFalpha receptors, whose expression in the developing CNS identifies oligodendrocyte progenitor cells (OPCs) and whose activation controls OPC proliferation, migration, survival, and maturation. PDGFRalpha-null mice die in utero, and OPCs that emerge before their demise have migration and proliferation defects and rapidly differentiate into postmitotic oligodendrocytes in vitro. OPCs from hemizygous mice also undergo precocious differentiation, indicating a role for PDGFRalpha gene dosage in timing OPC maturation. The rescue of PDGFRalpha-null OPCs with PDGFRalpha transgenes revealed specific roles for the phosphatidylinositol 3-kinase (PI3K) and phospholipase Cgamma (PLCgamma) pathways and a distinct ligand concentration dependence. Activation of the PI3K pathway is required for PDGFRalpha-induced migration, whereas activation of both PI3K and PLCgamma are required for PDGFRalpha-induced proliferation. For proliferation, PI3K activation is required at low ligand concentration, whereas PLCgamma is required at high signal strength. Dose-response studies further demonstrate that PDGFRalpha activates PI3K at low ligand concentrations, whereas PLCgamma is activated at high signal strength. Thus, PDGFRalpha signaling acts like a rheostat rather than generic ON switch, with signal strength dictating pathway activation during OPC maturation.

A specific requirement for PDGF-C in palate formation and PDGFR-alpha signaling.

PDGF-C is a member of the platelet-derived growth factor (PDGF) family, which signals through PDGF receptor (PDGFR) alphaalpha and alphabeta dimers. Here we show that Pdgfc(-/-) mice die in the perinatal period owing to feeding and respiratory difficulties associated with a complete cleft of the secondary palate. This phenotype was less severe than that of Pdgfra(-/-) embryos. Pdgfc(-/-) Pdgfa(-/-) embryos developed a cleft face, subepidermal blistering, deficiency of renal cortex mesenchyme, spina bifida and skeletal and vascular defects. Complete loss of function of both ligands, therefore, phenocopied the loss of PDGFR-alpha function, suggesting that both PDGF-A and PDGF-C signal through PDGFR-alpha to regulate the development of craniofacial structures, the neural tube and mesodermal organs. Our results also show that PDGF-C signaling is a new pathway in palatogenesis, different from, and independent of, those previously implicated.

IL-13 and IL-1beta promote lung fibroblast growth through coordinated up-regulation of PDGF-AA and PDGF-Ralpha.

Peribronchiolar fibrosis is a prominent feature of airway remodeling in asthma and involves fibroblast growth and collagen deposition. Interleukin-13 (IL-13), a T-helper 2 cytokine, is a key mediator of airway remodeling in asthma, yet the mechanism through which IL-13 promotes fibroblast growth has not been investigated. In this study, we show that IL-13 stimulates the mitogenesis of mouse, rat, and human lung fibroblasts through release of a soluble mitogen that we identified as PDGF-AA. The IL-13-induced growth of human lung fibroblasts was attenuated by an anti-PDGF-AA neutralizing antibody, and IL-13 stimulated human lung fibroblasts to secrete PDGF-AA. Fibroblasts derived from mouse embryos possessing the lethal Patch mutation, which lack the PDGF-Ralpha, showed no mitogenic response to IL-13. However, Patch cells did exhibit IL-13-induced STAT-6 phosphorylation. Stable transfection of the PDGF-Ralpha into Patch cells restored the growth response to PDGF-AA and IL-13. Through the use of lung fibroblasts from STAT-6-deficient mice, we showed that IL-13-induced PDGF-AA release is STAT-6 dependent, but PDGF-AA-induced growth is STAT-6 independent. Finally, we showed that IL-1beta enhanced IL-13-induced mitogenesis of rat lung fibroblasts through up-regulation of the PDGF-Ralpha. Our findings indicate that IL-13 acts in synergy with IL-1beta to stimulate growth by coordinately up-regulating PDGF-AA and the PDGF-Ralpha, respectively.

Cell autonomous requirement for PDGFRalpha in populations of cranial and cardiac neural crest cells.

Cardiac and cephalic neural crest cells (NCCs) are essential components of the craniofacial and aortic arch mesenchyme. Genetic disruption of the platelet-derived growth factor receptor alpha (PDGFRalpha) results in defects in multiple tissues in the mouse, including neural crest derivatives contributing to the frontonasal process and the aortic arch. Using chimeric analysis, we show that loss of the receptor in NCCs renders them inefficient at contributing to the cranial mesenchyme. Conditional gene ablation in NCCs results in neonatal lethality because of aortic arch defects and a severely cleft palate. The conotruncal defects are first observed at E11.5 and are consistent with aberrant NCC development in the third, fourth and sixth branchial arches, while the bone malformations present in the frontonasal process and skull coincide with defects of NCCs from the first to third branchial arches. Changes in cell proliferation, migration, or survival were not observed in PDGFRalpha NCC conditional embryos, suggesting that the PDGFRalpha may play a role in a later stage of NCC development. Our results demonstrate that the PDGFRalpha plays an essential, cell-autonomous role in the development of cardiac and cephalic NCCs and provides a model for the study of aberrant NCC development.

A human YAC transgene rescues craniofacial and neural tube development in PDGFRalpha knockout mice and uncovers a role for PDGFRalpha in prenatal lung growth.

The platelet-derived growth factor alpha-receptor (PDGFRalpha) plays a vital role in the development of vertebrate embryos, since mice lacking PDGFRalpha die in mid-gestation. PDGFRalpha is expressed in several types of migratory progenitor cells in the embryo including cranial neural crest cells, lung smooth muscle progenitors and oligodendrocyte progenitors. To study PDGFRalpha gene regulation and function during development, we generated transgenic mice by pronuclear injection of a 380 kb yeast artificial chromosome (YAC) containing the human PDGFRalpha gene. The YAC transgene was expressed in neural crest cells, rescued the profound craniofacial abnormalities and spina bifida observed in PDGFRalpha knockout mice and prolonged survival until birth. The ultimate cause of death was respiratory failure due to a defect in lung growth, stemming from failure of the transgene to be expressed correctly in lung smooth muscle progenitors. However, the YAC transgene was expressed faithfully in oligodendrocyte progenitors, which was not previously observed with plasmid-based transgenes containing only upstream PDGFRalpha control sequences. Our data illustrate the complexity of PDGFRalpha genetic control, provide clues to the location of critical regulatory elements and reveal a requirement for PDGF signalling in prenatal lung growth, which is distinct from the known requirement in postnatal alveogenesis. In addition, we found that the YAC transgene did not prolong survival of Patch mutant mice, indicating that genetic defects outside the PDGFRalpha locus contribute to the early embryonic lethality of Patch mice.