The Role of Chaperone-Mediated Autophagy in Bortezomib Resistant Multiple Myeloma.

Background: Multiple myeloma (MM) remains incurable despite high-dose chemotherapy, autologous stem cell transplants and novel agents. Even with the improved survival of MM patients treated with novel agents, including bortezomib (Bz), the therapeutic options in relapsed/refractory MM remain limited. The majority of MM patients eventually develop resistance to Bz, although the mechanisms of the resistance are poorly understood. Methods: Lysosomal associated membrane protein 2A (LAMP2A) mRNA and protein expression levels were assessed in ex vivo patient samples and a Bz-resistant MM cell line model by in real-rime PCR, western blotting and immunohistochemistry. In vitro modelling of chaperone-mediated autophagy (CMA) activity in response to ER stress were assessed by western blotting and confocal microscopy. The effects of CMA inhibition on MM cell viability and Bz sensitivity in MM cells were assessed by Annexin V/7AAD apoptosis assays using flow cytometry. Results: In this study, there is evidence that CMA, a chaperone-mediated protein degradation pathway, is upregulated in Bz-resistant MM and the inhibition of CMA sensitises resistant cells to Bz. The protein levels of LAMP2A, the rate-limiting factor of the CMA pathway, are significantly increased in MM patients resistant to Bz and within our Bz-resistant cell line model. Bz-resistant cell lines also possessed higher basal CMA activity than the Bz-sensitive parent cell line. In MM cell lines, CMA activity was upregulated in response to ER stress induced by Bz. The inhibition of CMA sensitises Bz-resistant cells to Bz and the combination of CMA inhibition and Bz in vitro had a more cytotoxic effect on myeloma cells than Bz alone. Conclusion: In summary, the upregulation of CMA is a potential mechanism of resistance to Bz and a novel target to overcome Bz-resistant MM.

An improved production and purification protocol for recombinant soluble human fibroblast activation protein alpha.

Fibroblast activation protein alpha (FAP) is a cell-surface expressed type II glycoprotein that has a unique proteolytic activity. FAP has active soluble forms that retain the extracellular portion but lack the transmembrane domain and cytoplasmic tail. FAP expression is normally very low in adult tissue but is highly expressed by activated fibroblasts in sites of tissue remodelling. Thus, FAP is a potential biomarker and pharmacological target in liver fibrosis, atherosclerosis, cardiac fibrosis, arthritis and cancer. Understanding the biological significance of FAP by investigating protein structure, interactions and activities requires reliable methods for the production and purification of abundant pure and stable protein. We describe an improved production and purification protocol for His6-tagged recombinant soluble human FAP. A modified baculovirus expression construct was generated using the pFastBac1 vector and the gp67 secretion signal to produce abundant active soluble recombinant human FAP (residues 27-760) in insect cells. The FAP purification protocol employed ammonium sulphate precipitation, ion exchange chromatography, immobilised metal affinity chromatography and ultrafiltration. High purity was achieved, as judged by gel electrophoresis and specific activity. The purified 82 kDa FAP protein was specifically inhibited by a FAP selective inhibitor, ARI-3099, and was inhibited by zinc with an IC50 of 25 µM. Our approach could be adopted for producing the soluble portions of other type II transmembrane glycoproteins to study their structure and function.

Regulatory innate lymphoid cells suppress innate immunity and reduce renal ischemia/reperfusion injury.

Innate lymphoid cells are a recently recognized group of immune cells with critical roles in tissue homeostasis and inflammation. Regulatory innate lymphoid cells are a newly identified subset of innate lymphoid cells, which play a suppressive role in the innate immune response, favoring the resolution of intestinal inflammation. However, the expression and role of regulatory innate lymphoid cells in kidney has not been reported. Here, we show that regulatory innate lymphoid cells are present in both human and mouse kidney, express similar surface markers and form a similar proportion of total kidney innate lymphoid cells. Regulatory innate lymphoid cells from kidney were expanded in vitro with a combination of IL-2, IL-7 and transforming growth factor-ß. These cells exhibited immunosuppressive effects on innate immune cells via secretion of IL-10 and transforming growth factor-ß. Moreover, treatment with IL-2/IL-2 antibody complexes (IL-2C) promoted expansion of regulatory innate lymphoid cells in vivo, and prevent renal ischemia/reperfusion injury in Rag-/- mice that lack adaptive immune cells including Tregs. Depletion of regulatory innate lymphoid cells with anti-CD25 antibody abolished the beneficial effects of IL-2C in the Rag-/- mice. Adoptive transfer of ex vivo expanded regulatory innate lymphoid cells improved renal function and attenuated histologic damage when given before or after induction of ischemia/reperfusion injury in association with reduction of neutrophil infiltration and induction of reparative M2 macrophages in kidney. Thus, our study shows that regulatory innate lymphoid cells suppress innate renal inflammation and ischemia/reperfusion injury.

Toward a Synergistic Operating Model for Westmead Research Hub Biobanks: A Questionnaire Study.

Standardization and sustainability are ideals within the biobanking world, and the demand for high-quality well-annotated specimens is growing just as rapidly as the ever-increasing precision and throughput of today's high-tech scientific methods. In the state of New South Wales (NSW) in Australia, the state government has allocated significant funding toward this requirement in recent years, with the launch of the NSW Health Statewide Biobank in central Sydney in 2017, and the introduction of the voluntary NSW Biobank Certification Program, and Consent Toolkit. For new and established biobanks, the influence of these new resources has been twofold: first they have provided valuable guidance for moving toward standardized practices and raising the bar for biobanking quality standards; second, they have brought to the forefront the challenges of sustainability and transitioning to a certification standard of biobanking. In Westmead, ∼20 km from Sydney's central business district, the Westmead Research Hub has responded to these challenges with a collaborative biobanking project initiated in 2015. As the site of almost 30 individual biobanks, and to inform a pilot project of central biobank services, a questionnaire was developed and administered to all of the biobanks. This article reports on the results from the questionnaire and the rationale for subsequent initiation of a core biobanking facility.

Standardisation of flow cytometry for whole blood immunophenotyping of islet transplant and transplant clinical trial recipients.

Understanding the immunological phenotype of transplant recipients is important to improve outcomes and develop new therapies. Immunophenotyping of whole peripheral blood (WPB) by flow cytometry is a rapid method to obtain large amounts of data relating to the outcomes of different transplant treatments with limited patient impact. Healthy individuals and patients with type 1 diabetes (T1D) enrolled in islet transplantation were recruited and WPB was collected. 46 fluorochrome-conjugated mouse-anti-human antibodies were used (43 of 46 antibodies were titrated). BD cytometer setup and tracking beads were used to characterize and adjust for cytometer performance. Antibody cocktails were pre-mixed <60 minutes before staining. Multicolour panels were designed based on fluorochrome brightness, antigen density, co-expression, and fluorochrome spillover into non-primary detectors in each panel on a 5 laser flow cytometer. WPB sample staining used 50-300 µl WPB for each panel and was performed within 2 hours of blood sample collection. Samples were acquired on a BD-LSRFortessa. The operating procedures, including specimen collection, antibody cocktails, staining protocol, flow-cytometer setup and data analysis, were standardized. The staining index of 43 antibodies and the spillover spreading matrix for each panel was calculated. The final concentrations for the 46 antibodies used was determined for staining of WPB samples. Absolute cell-count and 7 leukocyte profiling panels consisting of subsets and/or status of granulocytes, monocytes, dendritic, B, NK, and T cells including regulatory T cells (Tregs) and NKT were designed and established on a 5 laser BD-LSR Fortessa. 13 T1D patients, including 4 islet transplant recipients and 8 healthy controls, were evaluated. The ability to reproducibly measure immune subsets and immune-profiles of islet transplant patients up to 18 months post transplantation has been established as a tool to measure immune cell reconstitution after transplantation.

Dendritic cell-targeted CD40 DNA vaccine suppresses Th17 and ameliorates progression of experimental autoimmune glomerulonephritis.

The CD40-CD40L costimulatory pathway is critical for T cell activation in autoimmune disease. We have previously found that blocking the CD40-CD40L pathway using a dendritic cell-targeted CD40 DNA (DEC-CD40) vaccine prevented the development of Heymann nephritis. In this study, we explored the effect of a DEC-CD40 vaccine in the treatment of experimental autoimmune glomerulonephritis (EAG), an animal model of human Goodpasture's disease induced by antigen α3IV-NC1. DEC-CD40 vaccine given at week 3 and week 6 after 3IV-NC1 injection reduced kidney structural and functional injury significantly in EAG. DEC-CD40 vaccination suppressed Th17 cell numbers and Th17 immune responses in kidney and spleen, but did not alter Th1 cells number and responses. Serum derived from rats with DEC-CD40 vaccination suppressed Th17 differentiation, but not Th1 differentiation in vitro. Furthermore, B cell activation, driven by Th17 cytokines, was suppressed by serum from rats vaccinated with DEC-CD40. A DNA vaccine encoding CD40 and targeting dendritic cell, ameliorates kidney injury in both early and late stages in EAG rats, indicating DEC-CD40 vaccination has a therapeutic role in EAG. Its effect is associated with the reduction of Th17 differentiation and Th17-mediated B cell activation.

Flt3 inhibition alleviates chronic kidney disease by suppressing CD103+ dendritic cell-mediated T cell activation.

BACKGROUND: Chronic kidney disease (CKD) is a global public health problem, which lacks effective treatment. Previously, we have shown that CD103+ dendritic cells (DCs) are pathogenic in adriamycin nephropathy (AN), a model of human focal segmental glomerulosclerosis (FSGS). Fms-like tyrosine kinase 3 (Flt3) is a receptor that is expressed with high specificity on tissue resident CD103+ DCs. METHODS: To test the effect on CD103+ DCs and kidney injury of inhibition of Flt3, we used a selective Flt3 inhibitor (AC220) to treat mice with AN. RESULTS: Human CD141+ DCs, homologous to murine CD103+ DCs, were significantly increased in patients with FSGS. The number of kidney CD103+ DCs, but not CD103- DCs or plasmacytoid DCs, was significantly decreased in AN mice after AC220 administration. Treatment with AC220 significantly improved kidney function and reduced kidney injury and fibrosis in AN mice. AC220-treated AN mice had decreased levels of inflammatory cytokines and chemokines, tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, CCL2 and CCL5 and reduced kidney infiltration of CD4 T cells and CD8 T cells. The protective effect of AC220 was associated with its suppression of CD103+ DCs-mediated CD8 T cell proliferation and activation in AN mice. CONCLUSION: Flt3 inhibitor AC220 effectively reduced kidney injury in AN mice, suggesting that this inhibitor might be a useful pharmaceutical agent to treat CKD.

Potentiating Tissue-Resident Type 2 Innate Lymphoid Cells by IL-33 to Prevent Renal Ischemia-Reperfusion Injury.

The IL-33-type 2 innate lymphoid cell (ILC2) axis has an important role in tissue homeostasis, inflammation, and wound healing. However, the relative importance of this innate immune pathway for immunotherapy against inflammation and tissue damage remains unclear. Here, we show that treatment with recombinant mouse IL-33 prevented renal structural and functional injury and reduced mortality in mice subjected to ischemia-reperfusion injury (IRI). Compared with control-treated IRI mice, IL-33-treated IRI mice had increased levels of IL-4 and IL-13 in serum and kidney and more ILC2, regulatory T cells (Tregs), and anti-inflammatory (M2) macrophages. Depletion of ILC2, but not Tregs, substantially abolished the protective effect of IL-33 on renal IRI. Adoptive transfer of ex vivo-expanded ILC2 prevented renal injury in mice subjected to IRI. This protective effect associated with induction of M2 macrophages in kidney and required ILC2 production of amphiregulin. Treatment of mice with IL-33 or ILC2 after IRI was also renoprotective. Furthermore, in a humanized mouse model of renal IRI, treatment with human IL-33 or transfer of ex vivo-expanded human ILC2 ameliorated renal IRI. This study has uncovered a major protective role of the IL-33-ILC2 axis in renal IRI that could be potentiated as a therapeutic strategy.

Redirecting TGF-ß Signaling through the ß-Catenin/Foxo Complex Prevents Kidney Fibrosis.

TGF-ß is a key profibrotic factor, but targeting TGF-ß to prevent fibrosis also abolishes its protective anti-inflammatory effects. Here, we investigated the hypothesis that we can redirect TGF-ß signaling by preventing downstream profibrotic interaction of ß-catenin with T cell factor (TCF), thereby enhancing the interaction of ß-catenin with Foxo, a transcription factor that controls differentiation of TGF-ß induced regulatory T cells (iTregs), and thus, enhance anti-inflammatory effects of TGF-ß In iTregs derived from EL4 T cells treated with recombinant human TGF-ß1 (rhTGF-ß1) in vitro, inhibition of ß-catenin/TCF transcription with ICG-001 increased Foxp3 expression, interaction of ß-catenin and Foxo1, binding of Foxo1 to the Foxp3 promoter, and Foxo transcriptional activity. Moreover, the level of ß-catenin expression positively correlated with the level of Foxo1 binding to the Foxp3 promoter and Foxo transcriptional activity. T cell fate mapping in Foxp3gfp Ly5.1/5.2 mice revealed that coadministration of rhTGF-ß1 and ICG-001 further enhanced the expansion of iTregs and natural Tregs observed with rhTGF-ß1 treatment alone. Coadministration of rhTGF-ß1 with ICG-001 also increased the number of Tregs and reduced inflammation and fibrosis in the kidney fibrosis models of unilateral ureteric obstruction and ischemia-reperfusion injury. Notably, ICG-001 prevented the fibrosis in distant organs (lung and liver) caused by rhTGF-ß1. Together, our results show that diversion of ß-catenin from TCF- to Foxo-mediated transcription inhibits the ß-catenin/TCF-mediated profibrotic effects of TGF-ß while enhancing the ß-catenin/Foxo-mediated anti-inflammatory effects. Targeting ß-catenin/Foxo may be a novel therapeutic strategy in the treatment of fibrotic diseases that lead to organ failure.

Phenotypic and functional consequences of different isolation protocols on skin mononuclear phagocytes.

Mononuclear phagocytes are present in skin and mucosa and represent one of the first lines of defense against invading pathogens, which they detect via an array of pathogen-binding receptors expressed on their surface. However, their extraction from tissue is difficult, and the isolation technique used has functional consequences on the cells obtained. Here, we compare mononuclear phagocytes isolated from human skin using either enzymatic digestion or spontaneous migration. Cells isolated via enzymatic digestion are in an immature state, and all subsets are easily defined. However, cells isolated by spontaneous migration are in a mature state, and CD141 cross-presenting DCs (cDC1) are more difficult to define. Different pathogen-binding receptors are susceptible to cleavage by blends of collagenase, demonstrating that great care must be taken in choosing the correct enzyme blend to digest tissue if carrying out pathogen-interaction assays. Finally, we have optimized mononuclear phagocyte culture conditions to enhance their survival after liberation from the tissue.

CD103+ Dendritic Cells Elicit CD8+ T Cell Responses to Accelerate Kidney Injury in Adriamycin Nephropathy.

CD103(+) dendritic cells (DCs) in nonlymphoid organs exhibit two main functions: maintaining tolerance by induction of regulatory T cells and protecting against tissue infection through cross-presentation of foreign antigens to CD8(+) T cells. However, the role of CD103(+) DCs in kidney disease is unknown. In this study, we show that CD103(+) DCs are one of four subpopulations of renal mononuclear phagocytes in normal kidneys. CD103(+) DCs expressed DC-specific surface markers, transcription factors, and growth factor receptors and were found in the kidney cortex but not in the medulla. The number of kidney CD103(+) DCs was significantly higher in mice with adriamycin nephropathy (AN) than in normal mice, and depletion of CD103(+) DCs attenuated kidney injury in AN mice. In vitro, kidney CD103(+) DCs preferentially primed CD8(+) T cells and did not directly induce tubular epithelial cell apoptosis. Adoptive transfer of CD8(+) T cells significantly exacerbated kidney injury in AN SCID mice, whereas depletion of CD103(+) DCs in these mice impaired activation and proliferation of transfused CD8(+) T cells and prevented the exacerbation of kidney injury associated with this transfusion. In conclusion, kidney CD103(+) DCs display a pathogenic role in murine CKD via activation of CD8(+) T cells.

Flow cytometric analysis of circulating follicular helper T (Tfh) and follicular regulatory T (Tfr) populations in human blood.

Follicular helper T (Tfh) cells are the helper T-cell subset that localizes in germinal centers within secondary lymphoid organs. They support B cells to produce high-affinity antibodies and generate B-cell memory. By contrast, follicular regulatory T (Tfr) cells localize in germinal centers to suppress B-cell responses. Coordinately regulating antibody responses, Tfh and Tfr cells play a critical role for vaccination, infectious disease control, and the development of autoimmune diseases. Although bona fide Tfh and Tfr cells are hardly detected in human blood, circulating Tfh and Tfr memory cells in blood can be used to investigate their functions in health and disease. We have developed a comprehensive flow cytometric analysis to define different circulating Tfh and Tfr populations within CXCR5(+) CD4(+) T-cell population in human blood, based on numerous cell surface markers including CD25, CD127, CCR7, PD-1, CXCR3, and CCR6.

Renal F4/80+ CD11c+ mononuclear phagocytes display phenotypic and functional characteristics of macrophages in health and in adriamycin nephropathy.

Conventional markers of macrophages (Mфs) and dendritic cells (DCs) lack specificity and often overlap, leading to confusion and controversy regarding the precise function of these cells in kidney and other diseases. This study aimed to identify the phenotype and function of renal mononuclear phagocytes (rMPs) expressing key markers of both Mфs and DCs. F4/80(+)CD11c(+) cells accounted for 45% of total rMPs in normal kidneys and in those from mice with Adriamycin nephropathy (AN). Despite expression of the DC marker CD11c, these double-positive rMPs displayed the features of Mфs, including Mф-like morphology, high expression of CD68, CD204, and CD206, and high phagocytic ability but low antigen-presenting ability. F4/80(+)CD11c(+) cells were found in the cortex but not in the medulla of the kidney. In AN, F4/80(+)CD11c(+) cells displayed an M1 Mф phenotype with high expression of inflammatory mediators and costimulatory factors. Adoptive transfer of F4/80(+)CD11c(+) cells separated from diseased kidney aggravated renal injury in AN mice. Furthermore, adoptive transfer of common progenitors revealed that kidney F4/80(+)CD11c(+) cells were derived predominantly from monocytes, but not from pre-DCs. In conclusion, renal F4/80(+)CD11c(+) cells are a major subset of rMPs and display Mф-like phenotypic and functional characteristics in health and in AN.

A rare variant in human fibroblast activation protein associated with ER stress, loss of enzymatic function and loss of cell surface localisation.

Fibroblast activation protein (FAP) is a focus of interest as a potential cancer therapy target. This membrane bound protease possesses the unique catalytic activity of hydrolysis of the post-proline bond two or more residues from the N-terminus of substrates. FAP is highly expressed in activated fibroblastic cells in tumours, arthritis and fibrosis. A rare, novel, human polymorphism, C1088T, encoding Ser363 to Leu, occurring in the sixth blade of the ß propeller domain, was identified in a family. Both in primary human fibroblasts and in Ser363LeuFAP transfected cells, we showed that this single substitution ablates FAP dimerisation and causes loss of enzyme activity. Ser363LeuFAP was detectable only in endoplasmic reticulum (ER), in contrast to the distribution of wild-type FAP on the cell surface. The variant FAP showed decreased conformational antibody binding, consistent with an altered tertiary structure. Ser363LeuFAP expression was associated with upregulation of the ER chaperone BiP/GRP78, ER stress sensor ATF6, and the ER stress response target phospho-eIF2α, all indicators of ER stress. Proteasomal inhibition resulted in accumulation of Ser363LeuFAP, indicating the involvement of ER associated degradation (ERAD). Neither CHOP expression nor apoptosis was elevated, so ERAD is probably important for protecting Ser363LeuFAP expressing cells. These data on the first loss of function human FAP gene variant indicates that although the protein is vulnerable to an amino acid substitution in the ß-propeller domain, inactive, unfolded FAP can be tolerated by cells.

Regulatory T cells require renal antigen recognition through the TCR to protect against injury in nephritis.

Regulatory T cells (Treg) are important for maintaining immune homeostasis. Adoptive transfer of Tregs is protective in renal disease models in both immunocompetent and immunodeficient mice. However the involvement of TCR recognition of renal antigens remains to be clarified. To address this question, we made use of Tregs from the DO11.10 mouse (a TCR transgenic (Tg) mouse), that recognise the non-murine antigen Ovalbumin (OVA) and therefore are not activated by renal antigens. DO11.10 Tregs were assessed functionally in vitro and demonstrated equivalent suppression to WT BALB/c Tregs. Adriamycin Nephropathy (AN) was induced in mice which had been transfused with CD4+CD25+Tregs isolated from DO11.10 or BALB/c mice. To eliminate the memory/activation state as a cause of differences in activity, the protective capacity of DO11.10 Tregs pre-activated with OVA in vivo was assessed. Transfer of WT BALB/c Tregs significantly attenuated the development of AN with less glomerulosclerosis, tubular atrophy and macrophage infiltration as compared to AN mice without Treg transfer. However, mice receiving either naïve or pre-activated DO11.10 Tregs were not protected from AN. The lack of protection by DO11.10 Tregs was not due to failure to traffic to the affected kidney. These results suggest that antigen recognition in the kidney is important for Treg protection against injury.

Advances and issues in flow cytometric detection of immunophenotypic changes and genomic rearrangements in acute pediatric leukemia.

Flow cytometry with its rapidly increasing applications has been using to aid the diagnosis of hematological disorders for more than two decades. It is also the most commonly used technology in childhood leukaemia diagnosis, characterization, prognosis prediction and even in the decision making of targeted therapy. Leukemia cells can be recognized by virtue of unique cell marker combinations, visualized with monoclonal antibodies conjugated and detected by flow cytometry. Currently, such instruments allow the detection of eight or more markers by providing a comprehensive description of the leukemic cell phenotype to facilitate their identification, especially in detecting and monitoring of minimal residual disease (MRD) during treatment. Additionally, the flow cytometric DNA index (DI) can identify biclonality at diagnosis and distinguish persistent aneuploid leukemia during induction therapy, when the standard cytogenetic and morphologic techniques fail to do so. This review focuses on the latest advances and application issues about some of flow cytometric diagnostic and prognostic applications for acute pediatric leukemia.

High-throughput flow cytometry cell-based assay to detect antibodies to N-methyl-D-aspartate receptor or dopamine-2 receptor in human serum.

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

CD8+ regulatory T cells induced by T cell vaccination protect against autoimmune nephritis.

Autoreactive T cells play a pivotal role in the pathogenesis of autoimmune kidney disease. T cell vaccination (TCV) may limit autoimmune disease and induce CD8+ regulatory T cells (Tregs). We used Heymann nephritis (HN), a rat model of human membranous nephritis, to study the effects of TCV on autoimmune kidney disease. We harvested CD4+ T cells from renal tubular antigen (Fx1A) -immunized rats and activated these cells in vitro to express the MHC Class Ib molecule Qa-1. Vaccination of Lewis rats with these autoreactive Fx1A-induced T cells protected against HN, whereas control-primed T cells did not. Rats that underwent TCV had lower levels of proteinuria and serum creatinine and significantly less glomerulosclerosis, tubular damage, and interstitial infiltrates. Furthermore, these rats expressed less IFN-γ and IL-6 in splenocytes, whereas the numbers of Tregs and the expression of Foxp3 were unchanged. In vitro cytotoxicity assays showed CD8+ T cell-mediated elimination of Qa-1-expressing CD4+ T cells. In vivo, TCV abrogated the increase in Qa-1-expressing CXCR5+ TFH cells observed in HN compared with controls. Taken together, these results suggest that TCV protects against autoimmune kidney disease by targeting Qa-1-expressing autoreactive CD4+ cells.

Fibroblast activation protein and chronic liver disease.

Fibroblast activation protein (FAP) is the member of Dipeptidyl Peptidase IV (DPIV) gene family that is most similar to DPIV. Four members of this family, DPIV, FAP, DP8 and DP9 possess a rare catalytic activity, hydrolysis of a prolyl bond two residues from the substrate N terminus. Crystal structures show that the soluble form of FAP comprises two domains, an alpha/beta-hydrolase domain and an 8-blade beta-propeller domain. The interface between these two domains forms the catalytic pocket, and an opening for substrate access to the internal active site. The FAP homodimer is structurally very similar to DPIV but FAP glycoprotein expression is largely confined to mesenchymal cells in diseased and damaged tissue, notably the tissue remodelling region in chronically injured liver. FAP peptide substrates include denatured collagen and alpha2-antiplasmin. The functional roles of FAP in tumors and fibrotic tissue are not fully understood. This review places FAP in the context of chronic liver injury pathogenesis.

Fibroblast activation protein increases apoptosis, cell adhesion, and migration by the LX-2 human stellate cell line.

Injury and repair in chronic liver disease involve cell adhesion, migration, apoptosis, proliferation, and a wound healing response. In liver, fibroblast activation protein (FAP) has both collagenase and dipeptidyl peptidase IV (DPIV) activities and is expressed only by activated hepatic stellate cells (HSC) and myofibroblasts, which produce and degrade extracellular matrix (ECM). FAP was colocalized with collagen fibers, fibronectin, and collagen type I in human liver. FAP function was examined in vitro by expressing green fluorescent protein FAP fusion protein in cell lines cultured on collagen-I, fibronectin, and Matrigel. Glutamates at 203 and 204 as well as serine624 of FAP were essential for peptidase activity. Human embryonic kidney 293T cells overexpressing FAP showed reduced adhesion and migration. FAP overexpression in the human HSC line LX-2 caused increased cell adhesion and migration on ECM proteins as well as invasion across transwells in the absence or presence of transforming growth factor beta-1. FAP overexpression enhanced staurosporine streptomyces-stimulated apoptosis in both cell lines. Interestingly, the enzyme activity of FAP was not required for these functions. Overexpressing FAP increased the expression of matrix metalloproteinase-2 and CD44 and reduced integrin-beta1 expression in 293T cells, suggesting potential pathways of FAP-mediated impairment of cell adhesion and migration in this epithelial cell line. In conclusion, these findings further support a pro-fibrogenic role for FAP by indicating that, in addition to its enzymatic functions, FAP has important nonenzymatic functions that in chronic liver injury may facilitate tissue remodeling through FAP-mediated enhancement of HSC cell adhesion, migration, and apoptosis.