Leukemia inhibitory factor, a double-edged sword with therapeutic implications in human diseases.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine of the interleukin-6 (IL-6) superfamily. LIF was initially discovered as a factor to induce the differentiation of myeloid leukemia cells and thus inhibit their proliferation. Subsequent studies have highlighted the multi-functions of LIF under a wide variety of physiological and pathological conditions in a highly cell-, tissue-, and context-dependent manner. Emerging evidence has demonstrated that LIF plays an essential role in the stem cell niche, where it maintains the homeostasis and regeneration of multiple somatic tissues, including intestine, neuron, and muscle. Further, LIF exerts a crucial regulatory role in immunity and functions as a protective factor against many immunopathological diseases, such as infection, inflammatory bowel disease (IBD), and graft-verse-host disease (GVHD). It is worth noting that while LIF displays a tumor-suppressive function in leukemia, recent studies have highlighted the oncogenic role of LIF in many types of solid tumors, further demonstrating the complexities and context-dependent effects of LIF. In this review, we summarize the recent insights into the roles and mechanisms of LIF in stem cell homeostasis and regeneration, immunity, and cancer, and discuss the potential therapeutic options for human diseases by modulating LIF levels and functions.

Exogenous IL-4 shuts off pro-inflammation in neutrophils while stimulating anti-inflammation in macrophages to induce neutrophil phagocytosis following myocardial infarction.

INTRODUCTION: Macrophages and neutrophils are primary leukocytes involved in the inflammatory response to myocardial infarction (MI). While interleukin (IL)-4 is an in vitro anti-inflammatory stimulus, the MI myocardium does not express a considerable amount of IL-4 but does express IL4 receptors. We hypothesized that continuous exogenous IL-4 infusion starting 24 h after MI would promote a polarization switch in inflammatory cells towards a reparative phenotype. METHODS: C57BL/6J male mice (3-6 months of age) were subcutaneously infused with either saline (n = 17) or IL-4 (20 ng/g/day; n = 17) beginning 24 h after MI and evaluated at MI day 3. RESULTS: Macrophages and neutrophils were isolated ex vivo from the infarct region and examined. Exogenous IL-4 decreased pro-inflammatory Ccl3, Il12a, Tnfa, and Tgfb1 in neutrophils and increased anti-inflammatory Arg1 and Ym1 in macrophages (all p < .05). Tissue clearance by IL-4 treated neutrophils was not different, while selective phagocytosis of neutrophils doubled in IL-4 treated macrophages (p < .05). Of 24,339 genes examined by RNA-sequencing, 2042 genes were differentially expressed in macrophages from IL-4 stimulated infarct (all FDR p < .05). Pdgfc gene expression was ranked first, increasing 3-fold in macrophages stimulated with IL-4 (p = 1 × 10-9). Importantly, changes in macrophage physiology and transcriptome occurred in the absence of global LV effects. Bone marrow derived monocytes stimulated with mouse recombinant PDGF-CC protein (10 µg/ml) or PDGF-CC blocking antibody (200 ng/ml) did not change Arg1 or Ym1 expression, indicating the in vivo effect of IL-4 to stimulate macrophage anti-inflammatory gene expression was independent of PDGF-CC. CONCLUSIONS: Our results indicate that exogenous IL-4 promotes inflammation resolution by turning off pro-inflammation in neutrophils while stimulating anti-inflammation in macrophages to mediate removal of apoptotic neutrophils.

Identification and characterization of an alternative cancer-derived PD-L1 splice variant.

Therapeutic blockade of the PD-1/PD-L1 axis is recognized as an effective treatment for numerous cancer types. However, only a subset of patients respond to this treatment, warranting a greater understanding of the biological mechanisms driving immune evasion via PD-1/PD-L1 signaling and other T-cell suppressive pathways. We previously identified a head and neck squamous cell carcinoma with human papillomavirus integration in the PD-L1 locus upstream of the transmembrane domain-encoding region, suggesting expression of a truncated form of PD-L1 (Parfenov et al., Proc Natl Acad Sci USA 111(43):15544-15549, 2014). In this study, we extended this observation by performing a computational analysis of 33 other cancer types as well as human cancer cell lines, and identified additional PD-L1 isoforms with an exon 4 enrichment expressed in 20 cancers and human cancer cell lines. We demonstrate that cancer cell lines with high expression levels of exon 4-enriched PD-L1 generate a secreted form of PD-L1. Further biochemical studies of exon 4-enriched PD-L1 demonstrated that this form is secreted and maintains the capacity to bind PD-1 as well as to serve as a negative regulator on T cell function, as measured by inhibition of IL-2 and IFNg secretion. Overall, we have demonstrated that truncated forms of PD-L1 exist in numerous cancer types, and have validated that truncated PD-L1 can be secreted and negatively regulate T cell function.

Platelet-derived growth factor-D enables liver myofibroblasts to promote tumor lymphangiogenesis in cholangiocarcinoma.

BACKGROUND & AIMS: In cholangiocarcinoma, early metastatic spread via lymphatic vessels often precludes curative therapies. Cholangiocarcinoma invasiveness is fostered by an extensive stromal reaction, enriched in cancer-associated fibroblasts (CAFs) and lymphatic endothelial cells (LECs). Cholangiocarcinoma cells recruit and activate CAFs by secreting PDGF-D. Herein, we investigated the role of PDGF-D and liver myofibroblasts in promoting lymphangiogenesis in cholangiocarcinoma. METHODS: Human cholangiocarcinoma specimens were immunostained for podoplanin (LEC marker), α-SMA (CAF marker), VEGF-A, VEGF-C, and their cognate receptors (VEGFR2, VEGFR3). VEGF-A and VEGF-C secretion was evaluated in human fibroblasts obtained from primary sclerosing cholangitis explants. Using human LECs incubated with conditioned medium from PDGF-D-stimulated fibroblasts we assessed migration, 3D vascular assembly, transendothelial electric resistance and transendothelial migration of cholangiocarcinoma cells (EGI-1). We then studied the effects of selective CAF depletion induced by the BH3 mimetic navitoclax on LEC density and lymph node metastases in vivo. RESULTS: In cholangiocarcinoma specimens, CAFs and LECs were closely adjacent. CAFs expressed VEGF-A and VEGF-C, while LECs expressed VEGFR2 and VEGFR3. Upon PDGF-D stimulation, fibroblasts secreted increased levels of VEGF-C and VEGF-A. Fibroblasts, stimulated by PDGF-D induced LEC recruitment and 3D assembly, increased LEC monolayer permeability, and promoted transendothelial EGI-1 migration. These effects were all suppressed by the PDGFRß inhibitor, imatinib. In the rat model of cholangiocarcinoma, navitoclax-induced CAF depletion, markedly reduced lymphatic vascularization and reduced lymph node metastases. CONCLUSION: PDGF-D stimulates VEGF-C and VEGF-A production by fibroblasts, resulting in expansion of the lymphatic vasculature and tumor cell intravasation. This critical process in the early metastasis of cholangiocarcinoma may be blocked by inducing CAF apoptosis or by inhibiting the PDGF-D-induced axis. LAY SUMMARY: Cholangiocarcinoma is a highly malignant cancer affecting the biliary tree, which is characterized by a rich stromal reaction involving a dense population of cancer-associated fibroblasts that promote early metastatic spread. Herein, we show that cholangiocarcinoma-derived PDGF-D stimulates fibroblasts to secrete vascular growth factors. Thus, targeting fibroblasts or PDGF-D-induced signals may represent an effective tool to block tumor-associated lymphangiogenesis and reduce the invasiveness of cholangiocarcinoma.

Blocking IL-2 Signal In Vivo with an IL-2 Antagonist Reduces Tumor Growth through the Control of Regulatory T Cells.

IL-2 is critical for peripheral tolerance mediated by regulatory T (Treg) cells, which represent an obstacle for effective cancer immunotherapy. Although IL-2 is important for effector (E) T cell function, it has been hypothesized that therapies blocking IL-2 signals weaken Treg cell activity, promoting immune responses. This hypothesis has been partially tested using anti-IL-2 or anti-IL-2R Abs with antitumor effects that cannot be exclusively attributed to lack of IL-2 signaling in vivo. In this work, we pursued an alternative strategy to block IL-2 signaling in vivo, taking advantage of the trimeric structure of the IL-2R. We designed an IL-2 mutant that conserves the capacity to bind to the αß-chains of the IL-2R but not to the γc-chain, thus having a reduced signaling capacity. We show our IL-2 mutein inhibits IL-2 Treg cell-dependent differentiation and expansion. Moreover, treatment with IL-2 mutein reduces Treg cell numbers and impairs tumor growth in mice. A mathematical model was used to better understand the effect of the mutein on Treg and E T cells, suggesting suitable strategies to improve its design. Our results show that it is enough to transiently inhibit IL-2 signaling to bias E and Treg cell balance in vivo toward immunity.

Induction of dissociated cytokine profiles by ISRAA with selective critical involvement of ERK1/2 in its signaling functions.

The immune system-released activating agent (ISRAA) is an immune mediator activated as a result of a nerve stimulus initiated by immune challenge. We have previously demonstrated that ISRAA and tumor necrosis factor (TNF) receptor 1 (TNFR1) share an interspecies-conserved motif (72% homology) that induces the apoptosis and proliferation of human peripheral blood mononuclear cells (hPBMCs) in a dose-dependent manner. In the present study, cytokine profiles were examined in response to the stimulation of hPBMCs with ISRAA. Furthermore, the signaling pathways induced by ISRAA were mapped. The results revealed high measurable levels of TNF-α, interleukin (IL)-6, IL-8, IL-10 and interferon (IFN)-γ, but not IL-4, IL-17 (IL-17A) or transforming growth factor (TGF)-ß. The analysis of signaling pathways revealed the activation of extracellular-regulated protein kinase (ERK)1/2 as a downstream signal in the mitogen­activated protein kinase (MAPK) pathway during TNF­α and IL-6 production and apoptosis, but not during proliferation following stimulation with ISRAA by triggering the Fas-associated protein with death domain (FADD). STAT3 was found to be unphosphorylated in the ISRAA­stimulated hPBMCs, and STAT3 was ubiquitously expressed in unstimulated cells, suggesting that ISRAA has a protein inhibitor of activated STAT (PIAS)-like activity, by functioning as a negative regulator of the effects of STAT3 on the Janus kinase (JAK)/STAT pathway. The determination of the nature of cytokine responses together with the signaling pathways of cellular activity induced by ISRAA paves the way for the investigation of a potential target of ISRAA and for the development of novel therapeutic approaches for the treatment of immune-regulated disorders.

Inhibition of protein kinase C enhances angiogenesis induced by platelet-derived growth factor C in hyperglycemic endothelial cells.

BACKGROUND: Diabetes is a risk factor for the development of cardiovascular diseases with impaired angiogenesis. We have previously shown that platelet-derived growth factor C (PDGF-C) and its receptor, PDGF receptor α (PDGFR-α) were downregulated in ischemic limbs of diabetic mice, although the underlying mechanisms remained elusive. Protein kinase C (PKC) is a family of serine/threonine kinases and is known to be involved in angiogenesis. The purpose of this study is to elucidate the mechanisms of how PDGF-C/PDGFR-α axis is impaired in diabetes. METHODS: Human umbilical vein endothelial cells (HUVECs) and human cardiac microvascular endothelial cells (HMVECs) cultured in normoglycemic or hyperglycemic conditions were examined. We also examined the effects of PKC inhibition on the PDGF-C/PDGFR-α axis in endothelial cells exposed to hyperglycemia. RESULTS: Hyperglycemia inhibited proliferation and decreased viability of both HUVECs and HMVECs. Hyperglycemic endothelial cells exhibited decreased PDGFR-α expression both at messenger RNA (mRNA) and protein levels, while there was no significant change in expression of PDGF-C. We also found that expression of PKC-α, one of the PKC isoforms, was increased in hyperglycemic endothelial cells and that inhibition of PKC upregulated PDGFR-α expression in these cells. Phosphorylation of extracellular signal-regulated kinase (ERK) and Akt induced by PDGF-C was significantly attenuated in hyperglycemic endothelial cells, whereas inhibition of PKC effectively reversed these inhibitory effects. Moreover, inhibition of PKC also promoted angiogenesis induced by PDGF-C in hyperglycemic endothelial cells, which was not observed in vascular endothelial growth factor-A (VEGF-A)-induced angiogenesis. CONCLUSIONS: These findings suggest that downregulation of the PDGF-C/PDGFR-α axis is involved in impaired angiogenesis of hyperglycemia through upregulation of PKC. Targeting PKC to restore PDGF-C signaling might be a novel therapeutic strategy for the treatment of vascular complications in diabetes.

PDGF-D signaling in portal myofibroblasts and hepatic stellate cells proves identical to PDGF-B via both PDGF receptor type α and ß.

UNLABELLED: Platelet-derived growth factor-D (PDGF-D) is one member of PDGF growth factors and known to signal by binding to and activating its cognate receptor type ß (PDGFR-ß). Beside PDGF-B, PDGF-D is a potent growth factor for stellate cell growth and proliferation and therefore potentiates the extracellular matrix deposition in liver fibrogenesis. We aimed to explore the signaling and molecular mechanisms of PDGF-D in liver fibrogenesis using the primary liver portal myofibroblasts and hepatic stellate cells. Unexpectedly we found PDGF-D to bind to PDGFR-α, thus inducing receptor endocytosis and decreasing the amount of PDGFR-α significantly. PDGF-D activates PDGFR-α specific tyrosine 754 and -1018 phosphorylation and CrkII, the adaptor protein that is specifically recruited by activated PDGFR-α. As a novel finding we could also demonstrate that recombinant PDGFR-α-Fc chimera homodimer is able to bind PDGF-D and thus prevent PDGF-D signaling. PDGF-D does induce individual PDGFR-ß specific tyrosine phosphorylation similar to the PDGF-B. Additionally, PDGF-D enhances extracellular matrix accumulation comparable to the PDGF-B isoform. CONCLUSION: PDGF-D signaling in pMF and HSC is identical to that of PDGF-B by binding to both PDGFR-α and -ß.

Functional regulation of adipose-derived stem cells by PDGF-D.

Platelet-derived growth factor-D (PDGF-D) was recently identified, and acts as potent mitogen for mesenchymal cells. PDGF-D also induces cellular transformation and promotes tumor growth. However, the functional role of PDGF-D in adipose-derived stem cells (ASCs) has not been identified. Therefore, we primarily investigated the autocrine and paracrine roles of PDGF-D in this study. Furthermore, we identified the signaling pathways and the molecular mechanisms involved in PDGF-D-induced stimulation of ASCs. It is of interest that PDGF-B is not expressed, but PDGF-D and PDGF receptor-ß are expressed in ASCs. PDGF-D showed the strongest mitogenic effect on ASCs, and PDGF-D regulates the proliferation and migration of ASCs through the PI3K/Akt pathways. PDGF-D also increases the proliferation and migration of ASCs through generation of mitochondrial reactive oxygen species (mtROS) and mitochondrial fission. mtROS generation and fission were mediated by p66Shc phosphorylation, and BCL2-related protein A1 and Serpine peptidase inhibitor, clade E, member 1 mediated the proliferation and migration of ASCs. In addition, PDGF-D upregulated the mRNA expression of diverse growth factors such as vascular endothelial growth factor A, fibroblast growth factor 1 (FGF1), FGF5, leukemia inhibitory factor, inhibin, beta A, interleukin 11, and heparin-binding EGF-like growth factor. Therefore, the preconditioning of PDGF-D enhanced the hair-regenerative potential of ASCs. PDGF-D-induced growth factor expression was attenuated by a pharmacological inhibitor of mitogen-activated protein kinase pathway. In summary, PDGF-D is highly expressed by ASCs, where it acts as a potent mitogenic factor. PDGF-D also upregulates growth factor expression in ASCs. Therefore, PDGF-D can be considered a novel ASC stimulator, and used as a preconditioning agent before ASC transplantation.

An interspecies conserved motif of the mouse immune system-released activating agent (ISRAA) induces proliferative effects on human cells.

We have recently described an immune system-released activating agent (ISRAA) as a nervous system-induced factor that stimulates immune responses in the mouse spleen. However, the human ISRAA has not yet been identified. In this study, we examined the effects of the mouse ISRAA protein on human peripheral blood mononuclear cells (PBMCs), to observe if the biological activity of this molecule is consistent between the two different species. Mouse ISRAA demonstrated dose-dependent dualistic effects on human cells, as 5 µg exhibited positive apoptosis and 50 pg exhibited significant proliferation (P<0.05). Furthermore, immunosuppressed cells from patients undergoing immunosuppressive therapy demonstrated significant proliferation to 50 pg ISRAA (P<0.05). Studies to compare sequences in different species revealed a preserved motif, exhibiting 72% similarity with the interspecies conserved signal peptide motif of tumor necrosis factor receptor 1 (TNFR1). A mutant ISRAA lacking this motif was produced and tested for its biological effects. The mutant ISRAA demonstrated neither apoptotic nor proliferative effects compared with wild type. Therefore, an interspecies conserved domain of ISRAA constitutes the active site of the molecule, and its effects on immunocompromised cells should be investigated for future therapies in the treatment of immunosuppressive disorders.

Platelet-derived growth factor (PDGF)-C inhibits neuroretinal apoptosis in a murine model of focal retinal degeneration.

Platelet-derived growth factor (PDGF)-C is a member of the PDGF family and is critical for neuronal survival in the central nervous system. We studied the possible survival and antiapoptotic effects of PDGF-C on focal retinal lesions in Ccl2(-/-)/Cx3cr1(-/-) on C57BL/6N [Crb1(rd8)] (DKO rd8) background mice, a model for progressive and focal retinal degeneration. We found no difference in transcript and protein expression of PDGF-C in the retina between DKO rd8 mice and wild type (WT, C57BL/6N). Recombinant PDGF-CC protein (500 ng/eye) was injected intravitreally into the right eye of DKO rd8 mice with phosphate-buffered saline as controls into the left eye. The retinal effects of PDGF-C were assessed by fundoscopy, ocular histopathology, A2E levels, apoptotic molecule analysis, and direct flat mount retinal vascular labeling. We found that the PDGF-CC-treated eyes showed slower progression or attenuation of the focal retinal lesions, lesser photoreceptor and retinal pigment epithelial degeneration resulting in better-preserved photoreceptor structure. Lower expression of apoptotic molecules was detected in the PDGF-CC-treated eyes than in controls. In addition, no retinal neovascularization was observed after PDGF-CC treatment. Our results demonstrate that PDGF-C potently ameliorates photoreceptor degeneration via the suppression of apoptotic pathways without inducing retinal angiogenesis. The protective effects of PDGF-C suggest a novel alternative approach for potential age-related retinal degeneration treatment.

Diminished Th17 (not Th1) responses underlie multiple sclerosis disease abrogation after hematopoietic stem cell transplantation.

OBJECTIVE: To define changes in phenotype and functional responses of reconstituting T cells in patients with aggressive multiple sclerosis (MS) treated with ablative chemotherapy and autologous hematopoietic stem cell transplantation (HSCT). METHODS: Clinical and brain magnetic resonance imaging measures of disease activity were monitored serially in patients participating in the Canadian MS HSCT Study. Reconstitution kinetics of immune-cell subsets were determined by flow cytometry, whereas thymic function was assessed using T-cell receptor excision circle analyses as well as flow cytometry measurements of CD31+ recent thymic emigrants (RTEs). Functional assays were performed to track central nervous system-autoreactive antigen-specific T-cell responses, and the relative capacity to generate Th1, Th17, or Th1/17 T-cell responses. RESULTS: Complete abrogation of new clinical relapses and new focal inflammatory brain lesions throughout the 2 years of immune monitoring following treatment was associated with sustained decrease in naive T cells, in spite of restoration of both thymic function and release of RTEs during reconstitution. Re-emergence as well as in vivo expansion of autoreactive T cells to multiple myelin targets was evident in all patients studied. The reconstituted myelin-specific T cells exhibited the same Th1 and Th2 responses as preablation myelin-reactive T cells. In contrast, the post-therapy T-cell repertoire exhibited a significantly diminished capacity for Th17 responses. INTERPRETATION: Our results indicate that diminished Th17 and Th1/17 responses, rather than Th1 responses, are particularly relevant to the abrogation of new relapsing disease activity observed in this cohort of patients with aggressive MS following chemoablation and HSCT.

Growth arrest-specific protein 1 is a novel endogenous inhibitor of glomerular cell activation and proliferation.

Growth arrest-specific protein-1 (GAS1) is a GPI-anchored protein which is highly expressed in embryonic mouse fibroblasts and inhibits their proliferation. Glomerular mesangial cells release soluble GAS1 protein into the supernatant in vitro. Growth arrest led to GAS1 overexpression and increased release. Secretion involved disintegrin and metalloproteinase 10 and 17 as signified by inhibition experiments. Recombinant soluble GAS1 protein inhibited the proliferation of mesangial cells. Conversely, the induction of mesangial cell proliferation by PDGF-BB or -DD led to downregulation of GAS1 mRNA. Specific ligands of the PDGF α-receptor, PDGF-AA and -CC, had no effect. The GAS1 protein was localized in podocytes in kidneys from healthy rats. During the time course of mesangioproliferative glomerulonephritis in anti-Thy1.1-treated rats, glomerular GAS1 expression decreased prior to the onset of mesangial cell proliferation and increased at later stages during glomerular recovery. Finally, a plasmid expressing soluble GAS1 fused to an Fc fragment was systemically overexpressed in rats with mesangioproliferative glomerulonephritis. This ameliorated renal damage was indicated by decreased albuminuria and serum creatinine. Gas1/Fc-transfected rats also exhibited a reduction of the glomerular mesangial cell activation and proliferation. Thus, GAS1 is a novel endogenous inhibitor of glomerular mesangial cell proliferation and may be a novel therapeutic target in mesangioproliferative glomerular diseases.

Platelet derived growth factor-CC secreted by M2 macrophages induces alpha-smooth muscle actin expression by dermal and gingival fibroblasts.

Dermal wounds can heal detrimentally by formation of excess fibrosis or hypertrophic scarring. These phenomena are normally absent in the oral mucosa. Macrophages play an important role in wound repair, have a marked heterogeneity and are thought to contribute to fibrosis. To investigate to what extend macrophages are involved in the occurrence of fibrosis, the effect of differently activated macrophages on dermal and gingival fibroblasts was studied in vitro. Macrophages were differentiated into a classical (M1) or alternative (M2) phenotype, which was assessed by receptor expression (CD40/mannose receptor) and cytokine secretion (interleukin-4 and -12). Fibroblasts were exposed to these macrophages and/or conditioned medium (cm), and differentiation into α-SMA-expressing myofibroblasts was quantified. M2, but not M1 macrophages induced α-SMA expression in both dermal and gingival fibroblasts. Blocking of transforming growth factor-ß1 did not decrease the α-SMA expression mediated by M2 macrophages. It appeared that this induction was mediated by platelet derived growth factor-CC (PDGF-CC), produced by M2 macrophages. The expression and role of this growth factor was confirmed by ELISA, RT-PCR, and blocking experiments. Our results indicate that M2 macrophages are able to induce myofibroblast differentiation via production of PDGF-CC. Based on our findings we conclude that PDGF-CC may play a hitherto unknown role in the differentiation of myofibroblasts.

Discovery of small-molecule interleukin-2 inhibitors from a DNA-encoded chemical library.

Libraries of chemical compounds individually coupled to encoding DNA tags (DNA-encoded chemical libraries) hold promise to facilitate exceptionally efficient ligand discovery. We constructed a high-quality DNA-encoded chemical library comprising 30,000 drug-like compounds; this was screened in 170 different affinity capture experiments. High-throughput sequencing allowed the evaluation of 120 million DNA codes for a systematic analysis of selection strategies and statistically robust identification of binding molecules. Selections performed against the tumor-associated antigen carbonic anhydrase IX (CA IX) and the pro-inflammatory cytokine interleukin-2 (IL-2) yielded potent inhibitors with exquisite target specificity. The binding mode of the revealed pharmacophore against IL-2 was confirmed by molecular docking. Our findings suggest that DNA-encoded chemical libraries allow the facile identification of drug-like ligands principally to any protein of choice, including molecules capable of disrupting high-affinity protein-protein interactions.

A novel function for platelet-derived growth factor D: induction of osteoclastic differentiation for intraosseous tumor growth.

Although increasing evidence suggests a critical role for platelet-derived growth factor (PDGF) receptor ß (ß-PDGFR) signaling in prostate cancer (PCa) progression, the precise roles of ß-PDGFR and PDGF isoform-specific cell signaling have not been delineated. Recently, we identified the PDGF-D isoform as a ligand for ß-PDGFR in PCa and showed that PDGF-D is activated by serine protease-mediated proteolytic removal of the CUB domain in a two-step process, yielding first a hemidimer (HD) and then a growth factor domain dimer. Herein, we demonstrate that the expression of PDGF-D in human PCa LNCaP cells leads to enhanced bone tumor growth and bone responses in immunodeficient mice. Histopathological analyses of bone tumors generated by PDGF-D-expressing LNCaP cells (LNCaP-PDGF-D) revealed osteolytic and osteoblastic responses similar to those observed in human PCa bone metastases. Importantly, we discovered a novel function of PDGF-D in the regulation of osteoclast differentiation, independent of the RANKL/RANK signaling axis. Although both PDGF-B and -D were able to activate ß-PDGFR, only PDGF-D was able to induce osteoclastic differentiation in vitro, and upregulate the expression and nuclear translocation of nuclear factor of activated T cells 1, a master transcription factor for osteoclastogenesis. Taken together, these results reveal a new function of PDGF-D as a regulator of osteoclastic differentiation, an activity critical for the establishment of skeletal metastatic deposit in PCa patients.

In vivo anti- and pro-tumour activities of the TLR2 ligand FSL-1.

TLR ligands as Th1 inducers have been investigated as potential anti-tumour agents. However, few attempts have been made to investigate the anti-tumour activity of TLR ligands as Th2 inducers. This study, therefore, was carried out to determine whether the TLR2 ligand FSL-1 as a Th2 inducers affects the growth of a QRsP tumour, a fibrosarcoma derived from the C57BL/6 (TLR2(+/+)) mouse in vivo. Tumour volumes in TLR2(+/+) mice immunized with both FSL-1 and tumour-associated antigens were significantly smaller than those in control mice. Immunization with both FSL-1 and tumour-associated antigens increased the survival rate of TLR2(+/+) mice. However, surprisingly, immunization with FSL-1 alone significantly enhanced the growth of tumour. Both anti- and pro-tumour activities of FSL-1 were not observed in TLR2(-/-) mice. Immunization of both FSL-1 and tumour-associated antigens induced tumour-associated antigen-specific cytolytic T cells, antibody-dependent cell-mediated cytotoxicity of natural killer cells by production of the tumour-specific antibodies, tumour lysis by complement activation and reduction of the number of regulatory T cells in the draining lymph node. Immunization with FSL-1 alone increased the number of regulatory T cells in the draining lymph node, and in vivo administration of anti-CD25 antibody into mice abrogated the pro-tumour activity of FSL-1, suggesting that regulatory T cells are involved in the pro-tumour activity. This study demonstrated that FSL-1 exhibited TLR2-mediated anti- and pro-tumour activities when immunized with and without tumour-associated antigens, respectively.

PDGF-C mediates glomerular capillary repair.

Glomerular endothelial cell injury is a key component of a variety of diseases. Factors involved in glomerular endothelial cell repair are promising therapeutic agents for such diseases. Platelet-derived growth factor (PDGF)-C has pro-angiogenic properties; however, nothing is known about such functions in the kidney. We therefore investigated the consequences of either PDGF-C infusion or inhibition in rats with mesangioproliferative glomerulonephritis, which is accompanied by widespread glomerular endothelial cell damage. We also assessed the role of PDGF-C in a mouse model of thrombotic microangiopathy as well as in cultured glomerular endothelial cells. PDGF-C infusion in nephritic rats significantly reduced mesangiolysis and microaneurysm formation, whereas glomerular endothelial cell area and proliferation increased. PDGF-C infusion specifically up-regulated glomerular fibroblast growth factor-2 expression. In contrast, antagonism of PDGF-C in glomerulonephritis specifically reduced glomerular endothelial cell area and proliferation and increased mesangiolysis. Similarly, PDGF-C antagonism in murine thrombotic microangiopathy aggravated the disease and reduced glomerular endothelial area. In conditionally immortalized glomerular endothelial cells, PDGF-C was mitogenic and induced a 27-fold up-regulation of fibroblast growth factor-2 mRNA. PDGF-C also exerted indirect pro-angiogenic effects, since it induced endothelial cell mitogens and pro-angiogenic factors in mesangial cells and macrophages. These results identify PDGF-C as a novel, potent pro-angiogenic factor in the kidney that can accelerate capillary healing in experimental glomerulonephritis and thrombotic microangiopathy.

The anti-apoptotic protein G1P3 is overexpressed in psoriasis and regulated by the non-coding RNA, PRINS.

Psoriasis Susceptibility-Related RNA Gene Induced by Stress (PRINS) is a non-coding RNA overexpressed in lesional and non-lesional psoriatic epidermis and induced by stress. Its function in healthy and psoriatic skin is still not known. Here, we report that PRINS regulates G1P3, a gene with anti-apoptotic effects in keratinocytes. siRNA-mediated inhibition of PRINS gene resulted in altered cell morphology and gene expression alterations, as demonstrated in a microarray experiment. One of the genes regulated by PRINS ncRNA was G1P3, an interferon-inducible gene with anti-apoptotic effects in cancer cells. Interestingly, we found that G1P3 was 400-fold upregulated in hyperproliferative lesional and ninefold upregulated in non-lesional psoriatic epidermis compared to healthy epidermis. In vitro, G1P3 protein levels were highest in proliferating keratinocytes and siRNA-mediated downregulation of G1P3 resulted in increased cell apoptosis. These data indicate that G1P3 inhibits spontaneous keratinocyte apoptosis and hence its high expression in psoriatic skin may contribute to the development of psoriatic lesions. We hypothesize that the deregulation of the PRINS ncRNA may contribute to psoriasis and results in decreased sensitivity to spontaneous keratinocyte apoptosis via the regulation of G1P3.

Survival effect of PDGF-CC rescues neurons from apoptosis in both brain and retina by regulating GSK3beta phosphorylation.

Platelet-derived growth factor CC (PDGF-CC) is the third member of the PDGF family discovered after more than two decades of studies on the original members of the family, PDGF-AA and PDGF-BB. The biological function of PDGF-CC remains largely to be explored. We report a novel finding that PDGF-CC is a potent neuroprotective factor that acts by modulating glycogen synthase kinase 3beta (GSK3beta) activity. In several different animal models of neuronal injury, such as axotomy-induced neuronal death, neurotoxin-induced neuronal injury, 6-hydroxydopamine-induced Parkinson's dopaminergic neuronal death, and ischemia-induced stroke, PDGF-CC protein or gene delivery protected different types of neurons from apoptosis in both the retina and brain. On the other hand, loss-of-function assays using PDGF-C null mice, neutralizing antibody, or short hairpin RNA showed that PDGF-CC deficiency/inhibition exacerbated neuronal death in different neuronal tissues in vivo. Mechanistically, we revealed that the neuroprotective effect of PDGF-CC was achieved by regulating GSK3beta phosphorylation and expression. Our data demonstrate that PDGF-CC is critically required for neuronal survival and may potentially be used to treat neurodegenerative diseases. Inhibition of the PDGF-CC-PDGF receptor pathway for different clinical purposes should be conducted with caution to preserve normal neuronal functions.