Complement Factor B Expression Research Articles

Inhibition of the Alternative Complement Pathway May Cause Secretion of Factor B, Enabling an Early Detection of Pancreatic Cancer.

This study aims to elucidate the cellular mechanisms behind the secretion of complement factor B (CFB), known for its dual roles as an early biomarker for pancreatic ductal adenocarcinoma (PDAC) and as the initial substrate for the alternative complement pathway (ACP). Using parallel reaction monitoring analysis, we confirmed a consistent ∼2-fold increase in CFB expression in PDAC patients compared with that in both healthy donors (HD) and chronic pancreatitis (CP) patients. Elevated ACP activity was observed in CP and other benign conditions compared with that in HD and PDAC patients, suggesting a functional link between ACP and PDAC. Protein-protein interaction analyses involving key complement proteins and their regulatory factors were conducted using blood samples from PDAC patients and cultured cell lines. Our findings revealed a complex control system governing the ACP and its regulatory factors, including Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation, adrenomedullin (AM), and complement factor H (CFH). Particularly, AM emerged as a crucial player in CFB secretion, activating CFH and promoting its predominant binding to C3b over CFB. Mechanistically, our data suggest that the KRAS mutation stimulates AM expression, enhancing CFH activity in the fluid phase through binding. This heightened AM-CFH interaction conferred greater affinity for C3b over CFB, potentially suppressing the ACP cascade. This sequence of events likely culminated in the preferential release of ductal CFB into plasma during the early stages of PDAC. (Data set ID PXD047043.).

Inflammation Is More Sensitive than Cell Proliferation in Response to Rapamycin Treatment in Polycystic Kidney Disease

Introduction: It has been reported that rapamycin inhibited inflammation in renal interstitial diseases. We therefore hypothesized that rapamycin could attenuate inflammation in polycystic kidney disease (PKD). Methods: Han:SPRD rats were treated with rapamycin by daily gavage from 4 weeks to 12 weeks of age at the dosage of 0.5 mg/kg/day (low dose) or 1 mg/kg/day (high dose). WT9-12 human PKD cells were treated with various concentrations of rapamycin. Results: Two-kidney/total body weight ratio and cystic index in Cy/+ kidneys were significantly reduced with the treatment of low-dose rapamycin and further reduced by the treatment with high-dose rapamycin. However, the renal function of Cy/+ rats was equally improved by the treatment with either low-dose or high-dose rapamycin. The renal cell proliferation was significantly decreased in Cy/+ kidneys with the treatment of low-dose rapamycin and was further decreased with the treatment of high-dose rapamycin as examined by Ki67 staining. The phosphorylation of S6K in cystic kidneys was decreased by low-dose rapamycin and further decreased by high-dose rapamycin. Both low-dose and high-dose rapamycin treatment decreased macrophage infiltration and the expression of complement factor B (CFB), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-alpha (TNF-α) to a similar level. The expression of CFB, MCP-1, and TNF-α and phosphorylation of S6K were inhibited in WT9-12 cells treated with 10 nm rapamycin at 24 h and 48 h, respectively. Moreover, the phosphorylation of Akt was not increased by 1 nm and 10 nm of rapamycin and enhanced by 1 μm rapamycin treatment. Interestingly, WT9-12 cell proliferation could be inhibited by 1 μm rapamycin. Conclusion: Low dose of rapamycin could inhibit inflammation and protect renal function in PKD. Inflammation is more sensitive than cell proliferation in response to rapamycin treatment in PKD.

Functional variant rs12614 in CFB confers a low risk of IgA nephropathy by attenuating complement alternative pathway activation in Han Chinese.

Activation of the alternative pathway (AP) of complement is thought to play an important role in Immunoglobin A nephropathy (IgAN). Our previous study showed that rs4151657 within the complement factor B (CFB) gene increased the risk of IgAN. The protein encoded by the CFB gene is an initial factor that promotes AP activation. The aim of this study was to investigate whether other variants of CFB confer susceptibility to IgAN and elucidate their potential roles in AP activation. A total of 1,350 patients with IgAN and 1,420 healthy controls were enrolled and five tag single-nucleotide polymorphisms were selected for genotyping. The levels of key AP components, such as CFB, complement factor H and complement split product C3a, were measured by enzyme-linked immunosorbent assay. Molecular docking and molecular dynamic simulation were carried out to characterize the mutation of residues in the protein structure and the dynamic properties of wide type and mutation models of CFB protein. The allele-specific effect on CFB expression and its binding affinity to C3b were investigated through cell transfection and surface plasmon resonance analysis, respectively. We found that rs12614 significantly reduced the risk of IgAN (OR = 0.69, 95% CI = 0.52–0.91, P = 0.009), and the rs12614-T (R32W mutation) was correlated with lower CFB levels, higher serum C3 level, and less mesangial C3 deposition in patients with IgAN. The structural model showed that the R32W mutation reduced the structural stability of CFB protein. Furthermore, in vitro study revealed that rs12614-T decreased the expression of CFB and reduced its binding affinity to C3b by four-fold compared with rs12614-C. In conclusion, the rs12614-T in CFB was associated with low risk of IgAN probably by attenuating AP activation.

Complement factor B inhibitor LNP023 improves lupus nephritis in MRL/lpr mice

BackgroundLupus nephritis (LN) is the most common complication of systemic lupus erythematosus (SLE), and the abnormal activation of the alternative complement pathway is associated with the pathogenesis of LN. As an inhibitor of complement factor B (CFB) in the alternative pathway, LNP023 has been used in the treatment of a variety of renal diseases with abnormal complement system involvement, such as paroxysmal nocturnal hemoglobinuria, IgA nephropathy, and membranous nephropathy. The aim of our study was to explore whether LNP023 improved LN in MRL/lpr mice by inhibiting the activation of the alternative complement pathway. MethodsThe mice were divided into a normal control group (Normal group) (n = 6), MRL/lpr model group (n = 6), and LNP023 group (n = 6). The LNP023 group was administered LNP023 for 2 weeks by gavage; the MRL/lpr model group was administered saline for 2 weeks by gavage; and the Normal group was administered saline for 2 weeks by gavage. External signs, renal pathology, renal function, renal immune complex and complement deposition, serum anti-dsDNA, serum ANA concentration, and the expression of core complement factors in the alternative complement pathway were analyzed in the 3 groups of animals. The core targets of LNP023 in the treatment of LN were screened using network pharmacology. The pathogenicity of the core targets in LN was verified by analyzing the mRNA expression of the core targets in the peripheral blood mononuclear cells (PBMCs) of normal individuals, SLE patients, and LN patients. The mRNA and protein expression of core targets in the Normal group, MRL/lpr group, and LNP023 group were analyzed to verify whether LNP023 exerted it LN therapeutic effect through the regulation of core targets. ResultsCompared with the MRL/lpr group, the LNP023 group had reduced lupus-like signs, improved renal function, decreased serum anti-dsDNA and ANA concentrations, and reduced renal IgM, IgG, IgG1, C1q, C3, and C4 deposition. Renal pathology showed that LNP023 attenuated pathological damage in the kidneys of MRL/lpr mice. Compared with the MRL/lpr model group, the treatment group had no crescent formation, less immune deposition, no nuclear fragmentation, and less inflammatory cell infiltration. The expression of complement proteins C3, C3b, CR1, CFB, and C5b-9 in kidney tissues and liver was decreased, and the expression of C5 was increased. Network pharmacology screening indicated that AKT, TNF-α, MDM2, UBC, STST3, ESR1, and TP53 were core targets of LNP023 in the treatment of LN. Compared with that in the Normal group, the mRNA expression of the core target in the SLE and LN groups was different; compared with the MRL/lpr group, the LNP023 treatment group showed different mRNA and protein expression levels of AKT, TNF-α, and STST3. ConclusionLNP023 improves LN in MRL/lpr mice. The mechanism is as follows: LNP023 binds to CFB to inhibit the activation of the alternative complement pathway. LNP023 treatment for LN may also play a role in regulating the protein expression of AKT, TNF-α, and STST3.

The prognostic value of plasma complement factor B (CFB) in thyroid carcinoma

ABSTRACT Stromal and immune cells are major components of tumor microenvironment (TME) and affect the growth and development of thyroid carcinoma (THCA). However, data on the exact mechanisms that define the relationship between the TME and THCA remain scant. We calculated stromal and immune cells scores and the proportion of tumor-infiltrating immune cells (TICs) by CIBERSORT and ESTIMATE based on the THCA gene expression data from the Cancer Genome Atlas (TCGA). In addition, we evaluated differentially expressed genes (DEGs) from high- and low-score groups and performed functional enrichment analysis. Furthermore, our data show a significant correlation between plasma complement factor B (CFB) and PTC development and prognosis. Gene Set Enrichment Analysis (GSEA) demonstrated that the CFB was mainly enriched in immune response pathways. The expression of CFB was positively correlated with T cells CD8, Macrophages M1, Plasma cells, T cells CD4 memory activated, T cells follicular helper and T cells regulatory (Tregs), whereas negatively correlated with Eosinophils, Macrophages M0, Macrophages M2, Mast cells resting, T cells CD4 memory resting in the TME. Finally, the expression level of CFB was verified by other cohorts from Gene Expression Omnibus (GEO) database and quantitative Real-Time PCR (qRT-PCR) analyses, which was consistent with the results of bioinformatic analysis. Taken together, our data demonstrated that the CFB could be a prognostic marker for THCA and its expression influences the infiltration of immune cells.

Complement factor B in high glucose–induced podocyte injury and diabetic kidney disease

The role and mechanisms for upregulating complement factor B (CFB) expression in podocyte dysfunction in diabetic kidney disease (DKD) are not fully understood. Here, analyzing Gene Expression Omnibus GSE30528 data, we identified genes enriched in mTORC1 signaling, CFB, and complement alternative pathways in podocytes from patients with DKD. In mouse models, podocyte mTOR complex 1 (mTORC1) signaling activation was induced, while blockade of mTORC1 signaling reduced CFB upregulation, alternative complement pathway activation, and podocyte injury in the glomeruli. Knocking down CFB remarkably alleviated alternative complement pathway activation and DKD in diabetic mice. In cultured podocytes, high glucose treatment activated mTORC1 signaling, stimulated STAT1 phosphorylation, and upregulated CFB expression, while blockade of mTORC1 or STAT1 signaling abolished high glucose–upregulated CFB expression. Additionally, high glucose levels downregulated protein phosphatase 2Acα (PP2Acα) expression, while PP2Acα deficiency enhanced high glucose–induced mTORC1/STAT1 activation, CFB induction, and podocyte injury. Taken together, these findings uncover a mechanism by which CFB mediates podocyte injury in DKD.

Complement Factor B Mediates Ocular Angiogenesis through Regulating the VEGF Signaling Pathway.

Complement factor B (CFB), a 95-kDa protein, is a crucial catalytic element of the alternative pathway (AP) of complement. After binding of CFB to C3b, activation of the AP depends on the proteolytic cleavage of CFB by factor D to generate the C3 convertase (C3bBb). The C3 convertase contains the catalytic subunit of CFB (Bb), the enzymatic site for the cleavage of a new molecule of C3 into C3b. In addition to its role in activating the AP, CFB has been implicated in pathological ocular neovascularization, a common feature of several blinding eye diseases, however, with somewhat conflicting results. The focus of this study was to investigate the direct impact of CFB on ocular neovascularization in a tightly controlled environment. Using mouse models of laser-induced choroidal neovascularization (CNV) and oxygen-induced retinopathy (OIR), our study demonstrated an increase in CFB expression during pathological angiogenesis. Results from several in vitro and ex vivo functionality assays indicated a promoting effect of CFB in angiogenesis. Mechanistically, CFB exerts this pro-angiogenic effect by mediating the vascular endothelial growth factor (VEGF) signaling pathway. In summary, we demonstrate compelling evidence for the role of CFB in driving ocular angiogenesis in a VEGF-dependent manner. This work provides a framework for a more in-depth exploration of CFB-mediated effects in ocular angiogenesis in the future.

Low CFB expression is independently associated with poor overall and disease-free survival in patients with lung adenocarcinoma.

Complement factor B (CFB) serves a pivotal role in the alternative signaling pathway of the complement system and exerts a key role in the labelling of target particles, resulting from effective clearance of the target. The present study aimed to investigate the association between low expression levels of CFB and the clinical features and survival status of patients with lung adenocarcinoma (LUAD). Patient data were based on RNA-sequencing and clinical data from The Cancer Genome Atlas database. All patients were divided into two groups based on the median expression of CFB. Kaplan-Meier curve and univariate Cox regression analyses were used to investigate the association between CFB and survival status. Gene set enrichment analysis was used to examine the effects of CFB expression on signaling pathway impairment. Furthermore, reverse transcription-quantitative PCR (RT-qPCR) and western blotting were used to verify the relative expression levels of CFB in LUAD tissues. The data revealed that residual tumor classification, Karnofsky performance score and cancer stage were associated with overall survival, and that Karnofsky performance score and stage were associated with disease-free survival. The results demonstrated that high expression levels of CFB were associated with increased patient overall and disease-free survival according to both continuous and categorical models. The results of multivariate analysis identified that high expression levels of CFB were associated with increased overall and disease-free survival according to both the continuous model [hazard ratio (HR), 0.48; 95% confidence interval (95% CI), 0.25–0.93; P=0.029 for overall survival; HR, 0.29; 95% CI, 0.15–0.59; P=0.001 for disease-free survival] and the categorical model (HR, 0.46; 95% CI, 0.22–0.93; P=0.031 for overall survival; HR, 0.25; 95% CI, 0.12–0.55; P=0.001 for disease-free survival) after adjusting for corresponding covariates (residual tumour classification, Karnofsky performance score and stage). Furthermore, the results of both RT-qPCR and western blotting indicated that the relative mRNA and protein expression levels of CFB in lung tumor tissues were downregulated compared with those in adjacent non-tumor tissues. Collectively, the present results suggested that CFB expression was an independent predictor of overall and disease-free survival in patients with LUAD.

IL-1/IL-1R signaling induced by all-trans-retinal contributes to complement alternative pathway activation in retinal pigment epithelium.

The underlying mechanisms of complement activation in Stargardt disease type 1 (STGD1) and age-related macular degeneration (AMD) are not fully understood. Overaccumulation of all-trans-retinal (atRAL) has been proposed as the pathogenic factor in both diseases. By incubating retinal pigment epithelium (RPE) cells with atRAL, we showed that C5b-9 membrane attack complexes (MACs) were generated mainly through complement alternative pathway. An increase in complement factor B (CFB) expression as well as downregulation of complement regulatory proteins CD46, CD55, CD59, and CFH were observed in RPE cells after atRAL treatment. Furthermore, interleukin-1β production was provoked in both atRAL-treated RPE cells and microglia/macrophages. Coincubation of RPE cells with interleukin-1 receptor antagonist (IL1Ra) and atRAL ameliorated complement activation and downregulated CFB expression by attenuating both p38 and c-Jun N-terminal kinase (JNK) signaling pathways. Our findings demonstrate that atRAL induces an autocrine/paracrine IL-1/IL-1R signaling to promote complement alternative pathway activation in RPE cells and provide a novel perspective on the pathomechanism of macular degeneration.

Complement factor B knockdown by short hairpin RNA inhibits laser-induced choroidal neovascularization in rats.

To evaluate whether recombinant complement factor B (CFB) short hairpin RNA (shRNA) reduces laser-induced choroidal neovascularization (CNV) in rats. Laser-induced rat CNV model was established, and then the animals underwent fundus fluorescence angiography (FFA) and hematoxylin and eosin (HE) staining. On day 3 and 7 after photocoagulation, the expression of CFB and membrane attack complex (MAC) was detected by immunhischemistry. A recombinant CFB-shRNA plasmid was constructed. CFB and scrambled shRNA plasmids were intravenous injected into rats via the tail vein on the day of laser treatment, respectively. On day 7, the incidence of CNV was determined by FFA, and the expression of CFB and vascular endothelial growth factor (VEGF) in retinal pigment epithelium (RPE)/choroidal tissues was detected by immunhischemistry, Western blot and/or semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) in CFB and scrambled shRNA groups. The possible adverse effects of CFB-shRNA injection were assessed by transmission electron microscopy and electroretinography. FFA and HE results indicated that a laser-induced rat CNV model was successfully established on day 7 after photocoagulation. The expression of CFB and MAC was extremely weak in normal retina and choroid, and increased on day 3 after photocoagulation. However, it started to reduce on day 7. CFB shRNA plasmid was successfully constructed and induced CFB knockdown in the retinal and choroidal tissues. FFA showed CFB knockdown significantly inhibited incidence of CNV in rats. Moreover, CFB knockdown significantly inhibited the expression of VEGF in RPE/choroidal tissues. CFB shRNA caused no obvious side effects in eyes. CFB knockdown significantly inhibits the formation and development of CNV in vivo through reducing the expression of VEGF, which is a potential therapy target. The alternative pathway of complement activation plays an important role in CNV formation.

RA10.02: A NOVEL ROLE FOR THE COMPLEMENT CASCADE IN CHEMORADIATION THERAPY RESISTANT OESOPHAGEAL ADENOCARCINOMA

Abstract Background Resistance to chemoradiation therapy (CRT) in oesophageal adenocarcinoma (OAC) is an important clinical challenge. Elucidating underlying mechanisms of treatment resistance is crucial to improving survival rates. Methods OAC tumour biopsies were collected prior to neoadjuvant CRT. Tumour conditioned media was generated by culturing human explants for 24 h. Response to treatment was determined pathologically using the Mandard Grading System. C3, CFB and MCP-1 mRNA expression was assessed by qPCR. Secreted C3, C3a and MCP-1 were measured by ELISA. An isogenic model of radioresistant OAC was established by chronically irradiating OE33 cells with clinically-relevant doses of 2 Gy X-ray radiation. C5aR and C3aR expression was determined by flow cytometry. Results We demonstrate, for the first time, that the central factor of the complement cascade, complement component 3 (C3), is expressed in OAC tumours and is increased in pre-treatment OAC biopsies from patients (n = 13) who have a subsequent poor response to neoadjuvant CRT (P < 0.05). In addition, C3 is secreted from ex vivo human OAC tumour explants (n = 13) and correlates with levels of the monocyte chemoattractant MCP-1 (P < 0.008) and complement factor B (CFB) a component of the alternative pathway of complement activation (P < 0.0001). In vitro, radioresistant OE33 R cells had increased mRNA levels of C3 (P < 0.01), CFB (P < 0.05) and MCP-1 (P < 0.05) compared to radiosensitive OE33 P cells. OEE3 R cells expressed increased levels of secreted C3 and the anaphylatoxin C3a, compared to radiosensitive OE33P cells (both P < 0.001). Furthermore, we demonstrate for the first time that the anaphylatoxin receptor C5aR is expressed by OAC cells, suggesting that OAC cells can respond to complement anaphylatoxins. Conclusion This study highlights, for the first time, a novel role for the complement cascade in the resistance of OAC to CRT and highlights C3 as a novel predictive marker of response to CRT in OAC. Disclosure All authors have declared no conflicts of interest.

AICAR suppresses TNF-\u03b1-induced complement factor B in RPE cells

Age related macular degeneration is the leading cause of blindness in the developed world. Although its precise cause remains elusive, dysfunction of the retinal pigment epithelium (RPE) and dysregulation of complement have been implicated in its pathogenesis. The goal of this study was to evaluate the role of an AMP-dependent kinase (AMPK) activator, 5-aminoimidazole-4-carboxamide riboside (AICAR), on tumor necrosis factor alpha (TNF-α) induction of complement factor B (CFB) in RPE cells. We found that AICAR inhibited TNF-α-induced CFB expression in ARPE-19 and human primary RPE cells in a dose-dependent fashion. Treatment of cells with dipyridamole, which blocks AICAR cellular uptake abolished these effects. In contrast, the adenosine kinase inhibitor, 5-iodotubericidin, which inhibits the conversion of AICAR to the direct activator of AMPK, ZMP, did not reverse the effects on TNF-α-induced CFB expression, suggesting AMPK-independent effects. Indeed, knockout of AMPK in RPE cells using Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 did not abolish the inhibitory effects of AICAR on RPE CFB expression. Collectively, our results suggest that AICAR can suppress TNF-α-induced CFB expression in RPE cells in an AMPK-independent mechanism, and could be used as a therapeutic target in certain complement over-activation scenarios.

118 Complement components C3 and complement factor B promote growth of cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma (cSCC) is one of the most common metastatic skin cancers with increasing incidence. We examined the role of complement components C3 and complement factor B (CFB) in the growth of cSCC. Analysis of cSCC cell lines (n=8) and normal human epidermal keratinocytes (n=11) with quantitative real-time PCR and western blotting revealed upregulation of C3 and CFB expression in cSCC cells. Immunohistochemical staining revealed stronger tumor cell specific labeling for C3 and CFB in invasive cSCCs (n=71) and recessive dystrophic epidermolysis bullosa–associated cSCCs (n=11) than in cSCC in situ (n=69), actinic keratoses (n=63) and normal skin (n=5). Significant upregulation of C3 and CFB mRNA expression was noted in chemically induced mouse skin cSCCs, compared to benign papillomas, hyperplastic skin, and normal skin. The expression of C3 and CFB was higher in aggressive Ha-ras-transformed cell line (RT3) than in less tumorigenic HaCaT cell lines (HaCaT, A5, II-4). Knockdown of C3 and CFB expression inhibited migration and proliferation of cSCC cells and resulted in potent inhibition of ERK1/2 activation. Knockdown of C3 and CFB markedly inhibited growth of human cSCC xenograft tumors in vivo. These results provide evidence for the role of C3 and CFB in the development of cSCC and identify them as biomarkers and potential therapeutic targets in this metastatic skin cancer.

Complement Component C3 and Complement Factor B Promote Growth of Cutaneous Squamous Cell Carcinoma

Cutaneous squamous cell carcinoma (cSCC) is one of the most common metastatic skin cancers with increasing incidence. We examined the roles of complement component C3 and complement factor B (CFB) in the growth of cSCC. Analysis of cSCC cell lines (n=8) and normal human epidermal keratinocytes (n=11) with real-time quantitative PCR and Western blotting revealed up-regulation of C3 and CFB expression in cSCC cells. Immunohistochemical staining revealed stronger tumor cell-specific labeling for C3 and CFB in invasive cSCCs (n=71) and recessive dystrophic epidermolysis bullosa-associated cSCCs (n=11) than in cSCC in situ (n=69), actinic keratoses (n=63), and normal skin (n=5). Significant up-regulation of C3 and CFB mRNA expression was noted in chemically induced mouse cSCCs, compared to benign papillomas. Knockdown of C3 and CFB expression inhibited migration and proliferation of cSCC cells and resulted in potent inhibition of extracellular signal-regulated kinase 1/2 activation. Knockdown of C3 and CFB markedly inhibited growth of human cSCC xenograft tumors invivo. These results provide evidence for the roles of C3 and CFB in the development of cSCC and identify them as biomarkers and potential therapeutic targets in this metastatic skin cancer.

Resveratrol delays polycystic kidney disease progression through attenuation of nuclear factor κB-induced inflammation

Inflammation plays an important role in polycystic kidney disease (PKD). The current study aimed to examine the efficacy of the anti-inflammatory compound resveratrol in PKD and to investigate its underlying mechanism of action. Male Han:SPRD (Cy/+) rats with PKD were treated with 200 mg/kg/day resveratrol or vehicle by gavage for 5 weeks. Human autosomal dominant (AD) PKD cells, three-dimensional (3D) Madin-Darby canine kidney cells and zebrafish were treated with various concentrations of resveratrol or the nuclear factor κB (NF-κB) inhibitor QNZ. Resveratrol treatment reduced blood urea nitrogen levels and creatinine levels by 20 and 24%, respectively, and decreased two-kidney/total body weight ratio by 15% and cyst volume density by 24% in Cy/+ rats. The proliferation index and the macrophage infiltration index were reduced by 40 and 43%, respectively, in resveratrol-treated cystic kidneys. Resveratrol reduced the levels of the pro-inflammatory factors monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α) and complement factor B (CFB) in Cy/+ rat kidneys in parallel with the decreased activity of NF-κB (p50/p65). The activation of NF-κB and its correlation with pro-inflammatory factor expression were confirmed in human ADPKD cells and kidney tissues. Resveratrol and QNZ inhibited the expression of MCP-1, TNF-α and CFB and reduced NF-κB activity in ADPKD cells. Moreover, NF-κB blockage minimized the inhibition of inflammatory factor production by resveratrol treatment. Furthermore, resveratrol or QNZ inhibited cyst formation in the 3D cyst and zebrafish models. The NF-κB signaling pathway is activated and partly responsible for inflammation in polycystic kidney tissues. Targeting inflammation through resveratrol could be a new strategy for PKD treatment in the future.

The C-terminal tail of polycystin-1 regulates complement factor B expression by signal transducer and activator of transcription 1.

Inhibition of the overactivated alternative complement pathway in autosomal dominant polycystic kidney disease (ADPKD) retards disease progression in animal models; however, it remains unknown how complement factor B (CFB) is upregulated in ADPKD. Here, we showed that the overexpression of CFB in cystic kidneys is associated with increased JAK2/STAT1 activity and enhanced expression of the polycystin-1 C-terminal tail (PC1-CTT). Overexpression or blockage of STAT1 increased or decreased CFB expression and CFB promoter activity. Moreover, overexpression of PC1-CTT induced JAK2/STAT1 activation and CFB upregulation in renal tubular epithelial cells. Furthermore, PC1-CTT overexpression increased human CFB promoter activity, whereas dominant negative STAT1 plasmids or mutation of putative STAT1 responsive elements decreased PC1-CTT-induced CFB promoter activity. The effect of CFB on macrophage differentiation was tested on a mouse macrophage cell line. Bioactive CFB dose dependently promoted macrophage M2 phenotype conversion. In addition, conditioned media from renal epithelial cells promoted macrophage M2 phenotype conversion which was blocked by STAT1 inhibition in a dose-dependent manner. Conditioned media from PC1-CTT-transfected renal epithelial cells further promoted macrophage M2 phenotype conversion, which was suppressed by fludarabine or a CFB antibody. In addition, we show that NF-κB acts downstream of PC1-CTT and may partly mediate PC1-CTT-induced CFB expression. In conclusion, our study reveals possible mechanisms of CFB upregulation in ADPKD and a novel role of PC1-CTT in ADPKD-associated inflammation. Furthermore, our study suggests that targeting STAT1 may be a new strategy to prevent inflammation in the kidney of patients with ADPKD.

Abstract 3201: Complement component C3 and complement factor B regulate growth of cutaneous squamous cell carcinoma

Abstract Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer and its incidence is increasing globally. We have examined the role of complement component C3 and complement factor B (CFB) in the progression of cSCC. Significantly elevated mRNA levels for C3 and CFB were detected din cSCC cell lines (n = 8) compared to normal human epidermal keratinocytes (n = 11) by quantitative real-time reverse transcriptase-PCR. Increased production of C3 and CFB by cSCC cell lines was detected by western blotting. The mRNA levels for C3 and CFB were also markedly higher in cSCC tumors (n = 6) than in normal skin (n = 10). Tumor cell specific staining for C3 and CFB was detected in cSCC tumors in vivo by immunohistochemistry and the staining intensity was stronger in invasive cSCCs (n = 71) than in cSCC in situ (n = 69), premalignant epidermal lesions (actinic keratoses, n = 65) and normal skin (n = 5). Significant upregulation of C3 and CFB mRNA expression was also noted in chemically induced mouse skin cSCCs (n = 27) compared to benign papillomas (n = 17). The expression of C3 and CFB at mRNA and protein level was markedly higher in invasive tumorigenic Ha-ras-transformed HaCaT cell line than in benign tumorigenic ras-transformed HaCaT cells and parental non-tumorigenic HaCaT cells. The expression of both C3 and CFB was significantly upregulated by IFN-γ and TNF-α in cSCC cells. Knockdown of CFB and C3 by specific siRNAs inhibited migration of cSCC cells. Knockdown of CFB inhibited proliferation of cSCC cells and this was associated with potent inhibition of ERK1/2 activation. Knockdown of C3 and CFB also significantly inhibited growth of human cSCC xenograft tumors in vivo in SCID mice. These results provide evidence for the role of C3 and CFB in progression of cSCC and identify them as specific biomarkers and putative therapeutic targets for invasive cSCC. Citation Format: Pilvi Riihila, Mehdi Farshchian, Markku Kallajoki, Atte Kivisaari, Seppo Meri, Reidar Grenman, Ritva Heljasvaara, Taina Pihlajaniemi, Juha Peltonen, Sirkku Peltonen, Veli-Matti Kahari. Complement component C3 and complement factor B regulate growth of cutaneous squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3201. doi:10.1158/1538-7445.AM2015-3201

Identification of human complement factor B as a novel biomarker candidate for pancreatic ductal adenocarcinoma.

Pancreatic cancer (PC; pancreatic ductal adenocarcinoma) is characterized by significant morbidity and mortality worldwide. Although carbohydrate antigen (CA) 19-9 has been known as a PC biomarker, it is not commonly used for general screening because of its low sensitivity and specificity. Therefore, there is an urgent need to develop a new biomarker for PC diagnosis in the earlier stage of cancer. To search for a novel serologic PC biomarker, we carried out an integrated proteomic analysis for a total of 185 pooled or individual plasma from healthy donors and patients with five disease groups including chronic pancreatitis (CP), PC, and other cancers (e.g., hepatocellular carcinoma, cholangiocarcinoma, and gastric cancer) and identified complement factor b (CFB) as a candidate serologic biomarker for PC diagnosis. Immunoblot analysis of CFB revealed more than two times higher expression in plasma samples from PC patients compared with plasma from individuals without PC. Immunoprecipitation coupled to mass spectrometry analysis confirmed both molecular identity and higher expression of CFB in PC samples. CFB showed distinctly higher specificity than CA 19-9 for PC against other types of digestive cancers and in discriminating PC patients from non-PC patients (p < 0.0001). In receiver operator characteristic curve analysis, CFB showed an area under curve of 0.958 (95% CI: 0.956 to 0.959) compared with 0.833 (95% CI: 0.829 to 0.837) for CA 19-9. Furthermore, the Y-index of CFB was much higher than that of CA 19-9 (71.0 vs 50.4), suggesting that CFB outperforms CA 19-9 in discriminating PC from CP and other gastrointestinal cancers. This was further supported by immunoprecipitation and qRT-PCR assays showing higher expression of CFB in PC cell lines than in normal cell lines. A combination of CFB and CA 19-9 showed markedly improved sensitivity (90.1 vs 73.1%) over that of CFB alone in the diagnosis of PC against non-PC, with similar specificity (97.2 vs 97.9%). Thus, our results identify CFB as a novel serologic PC biomarker candidate and warrant further investigation into a large-scale validation and its role in molecular mechanism of pancreatic carcinogenesis.

Excessive activation of the alternative complement pathway in autosomal dominant polycystic kidney disease

The complement system is involved in many immune complex-mediated kidney diseases, yet its role in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD) has not been examined in detail. Screening of the glycoproteome of urine samples from ADPKD patients revealed that levels of complement factor B (CFB), serpin peptidase inhibitor, complement component 1 inhibitor (SERPING1) and complement component 9 (C9) increased, whereas complement component 1, r subcomponent-like (C1RL), CD55 and CD59 levels decreased with disease progression. Immunostaining and Western blot analysis confirmed the enhanced expression of CFB and C9 in cystic kidneys from ADPKD patients. Immunostaining also showed that the expressions of CFB and C9 in renal biopsy tissues from patients with other types of chronic kidney disease were lower than in tissues from ADPKD patients. The effect of the complement inhibitor rosmarinic acid (RMA) was evaluated in Pkd1(-/-) mice and Han:SPRD Cy/+ rats. Compared with vehicle-treated Pkd1(-/-) animals, RMA-treated mice had significantly lower serum creatinine (-50%) and blood urea nitrogen (-78%) levels, two kidneys/body weight ratio (-60%) and renal cystic index (-60%). Similar results were found in Cy/+ rats. Lower numbers of Ki67-positive nuclei and inflammatory cells and reduced fibrosis were observed in both animal models upon treatment with RMA. These results suggest that excessive activation of the alternative complement pathway is associated with ADPKD progression, probably mediated by cyst-lining epithelial cell proliferation, tubulointerstitial inflammatory cell infiltration and fibrosis. Targeting the complement system might represent a new therapeutic strategy for ADPKD.

Inhibition efficacy of CFB-siRNA on laser-induced choroidal neovascularization in rat

Background Choriodal neovascularization is an important ocular manifestation of angiogenesis in eyes,which derives from the choroid capillaries.Recent studies have found that complement activation is playing a key role in the laser-induced CNV.Because of the key position of CFB in the alternative pathway,bytargeting CFB and blocking the alternative pathway may provide an approach to observe the role of this alternative pathway in the generation of CNV. Objective This study was to investigate the inhibitory effect of reconstructed complement factor B (CFB)-small interfering ribonucleic acid (siRNA) on choroidal neovascularization (CNV)and its mechanism. Methods Experimental CNV was induced by laser photocoagulation in 96 eyes of 48 clean Brown Norway rats.The rats were randomly divided into 4 groups.25,50 and 75 μg B factor siRNA were injected via caudal vein on 1 day,3,5 days after photocoagulation in different dose groups,and normal saline solution was injected at the same way in experimental control group.Other 12 normal rats were used as blank control group.Fundus fluorescein angiography(FFA) was performed on 3,7,14,21,28 days after injection of CFB-siRNA and CNV was scored.The expressions of vascular endothelial growth factor(VEGF) and factor Ⅷ in choroid were detected by immunochemistry.The expressions of CFB-siRNA,VEGF,transforming growth factor β2( TGF-β2 )proteins in choroid were determined using immunochemistry in 7,14,21,28 days,and the expressions of mRNA of CFB-siRNA,VEGF,TGF-β2 were examined by reverse transcription polymerase chain reaction(RT-PCR). Results FFA revealed that the CNV rates in various doses of CFB-siRNA groups were significant lower than those of experimental control group in various time points(P＜0.05),and those in 75 μg B factor siRNA were decreased in comparison with 25 μg B factor siRNA (P＜0.05).Immunochemistry showed that the intensities of the VEGF and factor Ⅶ expression in various doses of CFB-siRNA groups were weaker than the blank control group ( P ＜ 0.05 ).Compared with the control group,the expression of CFB reduced in 7 days,and then approached to the level near the control group.Fourteen to twenty-one days after injection of CFB-siRNA,VEGF and TGF-β2 depressions in different doses of CFB-siRNA groups were lower than blank control group( P＜0.05 ).CFB expression in choroid showed the lower levels in CFB-siRNA injection group compared with blank control group in from 7 through 21 days (P＜0.05).RT-PCR displayed the gradual increase of CFB mRNA and curve-like changes of VEGF and TGF-β2 with time prolong. Conclusions Recombinated CFB-siRNA can effectively inhibit laser-induced CNV by down-regulating the expression of VEGF and factor Ⅷ.Alternative pathway of complement plays an important role in the production of CNV. Key words: Choroidal neovascularization/management; Gene therapy; Complement factor B; Small interfering RNA