Expression of cathepsins B, D and K in thymic epithelial tumours.

AIM: Cathepsins are proteases that regulate a wide range of physiological processes, including protein turnover, cell signalling and antigen presentation. Recent studies have shown that cathepsins are highly upregulated in many types of tumours. Of the 15 cathepsins in humans, cathepsins V and S are abundantly expressed in the thymus, and we previously showed that the immunostaining of these cathepsins could serve as diagnostic markers for thymic epithelial tumours. However, little is known about the expression of other cathepsins in thymic epithelial tumours. To determine the diagnostic implications of cathepsins, we performed immunohistochemical analysis of cathepsin B (CTB), cathepsin D (CTD) and cathepsin K (CTK), all of which have been reported to correlate with the progression of squamous cell carcinoma. METHODS: The association between cathepsin expression and clinicopathological features was evaluated in 122 cases of thymoma and thymic carcinoma. RESULTS: CTB and CTD were frequently expressed in type A and type AB thymomas. In contrast, CTB and CTD were significantly less common in type B thymomas than in type A or AB thymomas. In type AB thymomas, the expression of CTB correlated with histological features, and was found predominantly in the type A component. Notably, CTK was expressed most commonly in thymic carcinomas, and patients who died of the disease showed increased expression of CTK. CONCLUSIONS: The expression of CTB and CTD correlated with the histological subtype of thymoma. In addition, the expression of CTK appears to be useful for the diagnosis of thymic carcinomas and as a prognostic marker.

Evidence for lysosomal biogenesis proteome defect and impaired autophagy in preeclampsia.

The etiology of preeclampsia (PE), a serious pregnancy complication, remains an enigma. We have demonstrated that proteinopathy, a pathologic feature of neurodegenerative diseases, is a key observation in the placenta and serum from PE patients. We hypothesize that the macroautophagy/autophagy machinery that mediates degradation of aggregated proteins and damaged organelles is impaired in PE. Here, we show that TFEB (transcription factor EB), a master transcriptional regulator of lysosomal biogenesis, and its regulated proteins, LAMP1, LAMP2, and CTSD (cathepsin D), were dysregulated in the placenta from early and late onset PE deliveries. Primary human trophoblasts and immortalized extravillous trophoblasts (EVTs) showed reduced TFEB expression and nuclear translocation as well as lysosomal protein content in response to hypoxia. Hypoxia-exposed trophoblasts also showed decreased PPP3/calcineurin phosphatase activity and increased XPO1/CRM1 (exportin 1), events that inhibit TFEB nuclear translocation. These proteins were also dysregulated in the PE placenta. These results are supported by observed lysosomal ultrastructural defects with decreased number of autolysosomes in hypoxia-treated primary human trophoblasts. Autophagy-deficient human EVTs exhibited poor TFEB nuclear translocation, reduced lysosomal protein expression and function, and increased MTORC1 activity. Sera from PE patients induced these features and protein aggregation in EVTs. Importantly, trophoblast-specific conditional atg7 knockout mice exhibited reduced TFEB expression with increased deposition of protein aggregates in the placenta. These results provide compelling evidence for a regulatory link between accumulation of protein aggregates and TFEB-mediated impaired lysosomal biogenesis and autophagy in the placenta of PE patients. Abbreviation:atg7: autophagy related 7; CTSD: cathepsin D; ER: endoplasmic reticulum; EVTs: extravillous trophoblasts; KRT7: keratin 7; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; mSt: mStrawberry; MTORC1: mechanistic target of rapamycin complex 1; NP: normal pregnancy; NPS: normal pregnancy serum; PE: preeclampsia; PES: preeclampsia serum; p-RPS6KB: phosphorylated ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TFEB: transcription factor EB; XPO1/CRM1: exportin 1.

Schistosoma mansoni cathepsin D1: Biochemical and biophysical characterization of the recombinant enzyme expressed in HEK293T cells.

Schistosomes express a variety of aspartyl proteases (APs) with distinct roles in the helminth pathophysiology, among which degradation of host haemoglobin is key, since it is the main amino acid source for these parasites. A cathepsin D-like AP from Schistosoma mansoni (SmCD1) has been used as a model enzyme for vaccine and drug development studies in schistosomes and yet a reliable expression system for readily producing the recombinant enzyme in high yield has not been reported. To contribute to further advancing the knowledge about this valuable antischistosomal target, we developed a transient expression system in HEK 293T mammalian cells and performed a biochemical and biophysical characterization of the recombinant enzyme (rSmCD1). It was possible to express a recombinant C-terminal truncated form of SmCD1 (rSmCD1ΔCT) and purify it with high yield (16 mg/L) from the culture supernatant. When analysed by Size-Exclusion Chromatography and multi-angle laser light scattering, rSmCD1ΔCT behaved as a dimer at neutral pH, which is unusual for cathepsins D, turning into a monomer after acidification of the medium. Through analytical ultrancentrifugation, the dimer was confirmed for free rSmCD1ΔCT in solution as well as stabilization of the monomer during interaction with pepstatin. The mammalian cell expression system used here was able to produce rSmCD1ΔCT with high yields allowing for the first time the characterization of important kinetic parameters as well as initial description of its biophysical properties.

Is digestive cathepsin D the rule in decapod crustaceans?

Cathepsin D is an aspartic endopetidase with typical characteristics of lysosomal enzymes. Cathepsin D activity has been reported in the gastric fluid of clawed lobsters where it acts as an extracellular digestive enzyme. Here we investigate whether cathepsin D is unique in clawed lobsters or, instead, common in decapod crustaceans. Eleven species of decapods belonging to six infraorders were tested for cathepsin D activity in the midgut gland, the muscle tissue, the gills, and when technically possible, in the gastric fluid. Cathepsin D activity was present in the midgut gland of all 11 species and in the gastric fluid from the seven species from which samples could be taken. All sampled species showed higher activities in the midgut glands than in non-digestive organs and the activity was highest in the clawed lobster. Cathepsin D mRNA was obtained from tissue samples of midgut gland, muscle, and gills. Analyses of deduced amino acid sequence confirmed molecular features of lysosomal cathepsin D and revealed high similarity between the enzymes from Astacidea and Caridea on one side, and the enzymes from Penaeoidea, Anomura, and Brachyura on the other side. Our results support the presence of cathepsin D activity in the midgut glands and in the gastric fluids of several decapod species suggesting an extracellular function of this lysosomal enzyme. We discuss whether cathepsin D may derive from the lysosomal-like vacuoles of the midgut gland B-cells and is released into the gastric lumen upon secretion by these cells.

Immunohistochemical Expression of Cathepsin D in Primary and Recurrent Squamous Cell Carcinoma.

AIM: The aim of this study is to analyze and compare the immunohistochemical expression of cathepsin B in primary oral squamous cell carcinoma (OSCC) and recurrent OSCC. MATERIALS AND METHODS: A total of 50 cases were studied immunohistochemically for rabbit polyclonal antihuman cathepsin D expression. A total of 10 cases of breast carcinoma were taken as positive controls. Immunohistochemical staining was performed using labeled streptavidin-biotin technique. RESULTS: All the 45 cases of OSCC, both primary and recurrent cases included, showed varying grades of cathepsin D immu-noreactivity. Statistical significance at 5% level was observed in cathepsin D expression between the different grades of well, moderate, and poorly differentiated primary squamous cell carcinomas. In the comparison of cathepsin D staining intensity among primary squamous cell carcinomas with and without recurrence, a statistical significance between the groups was observed when the p-value was at 10%, but the same comparison was not significant when the p-value was at 5%. CONCLUSION: Cathepsin D expression in primary squamous cell carcinomas with recurrences was very variable as compared with primary squamous cell carcinomas without recurrences. Comparison of cathepsin D expression in primary with their recurrent counterparts showed mostly similar intensity of expression in recurrent carcinomas, thus suggesting its limited usefulness in predicting recurrence. CLINICAL SIGNIFICANCE: Although cathepsin D might have shown limited usefulness in predicting cancer recurrence, it, however, is a proven valuable tool to detect the aggressiveness of various other tumors, and if corroborated with a larger sample may hold the key to early, more effective, and more specific treatment modalities for cases of oral cancer also.

Increased expression of transcription factor EB (TFEB) is associated with autophagy, migratory phenotype and poor prognosis in non-small cell lung cancer.

OBJECTIVES: We investigated the role of lysosomal biogenesis and hydrolase activity in the clinical behavior and postoperative outcome of lung cancer. MATERIALS AND METHODS: Using immunohistochemistry we investigated the expression of the transcription factor EB (TFEB) which orchestrates lysosomal biogenesis, the lysosome membrane protein LAMP2a and of the lysosomal hydrolase cathepsin D in a series of 98 non-small cell lung carcinomas (NSCLC) treated with surgery alone. In vitro experiments with the A549 and H1299 lung cancer cell lines were also performed. RESULTS: Overexpression of TFEB, LAMP2a and Cathepsin D was noted in 47/98 (47.9%), 43/98 (43.9%) and 39/98 (39.8%) cases, respectively, and were significantly correlated with each other and with adenocarcinomas. High LAMP2a was related to high histology grade. Linear regression analysis confirmed significant association of TFEB with BNIP3 (p=0.0003, r=0.35) and LC3A with LAMP2a expression (p=0.0002, r=0.37). An inverse association of Cathepsin D expression with stone-like structures (SLS) was recorded (p=0.02, r=0.22). On univariate analysis all three lyososomal variables were associated with poor prognosis (p=0.05, 0.04 and 0.01, for TFEB, Cathepsin D and LAMP2a, respectively). Multivariate analysis showed that the SLS number (p=0.0001, HR5.37), Cathepsin D expression (p=0.01, HR=2.2) and stage (p=0.01, HR=1.5) were independent prognostic variables. Silencing of TFEB with siRNAs in the A549 and H1299 lung cancer cell lines did not affect proliferation but resulted in reduced migration ability. CONCLUSION: Lysosomal biogenesis is linked to autophagosomal protein expression in NSCLC and characterizes subgroups of high risk patients after complete surgical lung tumor resection.

Increased cathepsin D protein expression is a biomarker for osteosarcomas, pulmonary metastases and other bone malignancies.

Cancer proteomics provide a powerful approach to identify biomarkers for personalized medicine. Particularly, biomarkers for early detection, prognosis and therapeutic intervention of bone cancers, especially osteosarcomas, are missing. Initially, we compared two-dimensional gel electrophoresis (2-DE)-based protein expression pattern between cell lines of fetal osteoblasts, osteosarcoma and pulmonary metastasis derived from osteosarcoma. Two independent statistical analyses by means of PDQuest® and SameSpot® software revealed a common set of 34 differentially expressed protein spots (p < 0.05). 17 Proteins were identified by mass spectrometry and subjected to Ingenuity Pathway Analysis resulting in one high-ranked network associated with Gene Expression, Cell Death and Cell-To-Cell Signaling and Interaction. Ran/TC4-binding protein (RANBP1) and Cathepsin D (CTSD) were further validated by Western Blot in cell lines while the latter one showed higher expression differences also in cytospins and in clinical samples using tissue microarrays comprising osteosarcomas, metastases, other bone malignancies, and control tissues. The results show that protein expression patterns distinguish fetal osteoblasts from osteosarcomas, pulmonary metastases, and other bone diseases with relevant sensitivities between 55.56% and 100% at ≥87.50% specificity. Particularly, CTSD was validated in clinical material and could thus serve as a new biomarker for bone malignancies and potentially guide individualized treatment regimes.

BCM-95 and (2-hydroxypropyl)-ß-cyclodextrin reverse autophagy dysfunction and deplete stored lipids in Sap C-deficient fibroblasts.

Saposin (Sap) C deficiency is a rare variant form of Gaucher disease caused by impaired Sap C expression or accelerated degradation, and associated with accumulation of glucosylceramide and other lipids in the endo/lysosomal compartment. No effective therapies are currently available for the treatment of Sap C deficiency. We previously reported that a reduced amount and enzymatic activity of cathepsin (Cath) B and Cath D, and defective autophagy occur in Sap C-deficient fibroblasts. Here, we explored the use of two compounds, BCM-95, a curcumin derivative, and (2-hydroxypropyl)-ß-cyclodextrin (HP-ß-CD), to improve lysosomal function of Sap C-deficient fibroblasts. Immunofluorescence and biochemical studies documented that each compound promotes an increase of the expression levels and activities of Cath B and Cath D, and efficient clearance of cholesterol (Chol) and ceramide (Cer) in lysosomes. We provide evidence that BCM-95 and HP-ß-CD enhance lysosomal function promoting autophagic clearance capacity and lysosome reformation. Our findings suggest a novel pharmacological approach to Sap C deficiency directed to treat major secondary pathological aspects in this disorder.

Plasma asymmetric and symmetric dimethylarginine in a rat model of endothelial dysfunction induced by acute hyperhomocysteinemia.

Hyperhomocysteinemia induces vascular endothelial dysfunction, an early hallmark of atherogenesis. While higher levels of circulating asymmetric dimethylarginine (ADMA) and symmetric dimethyl arginine (SDMA), endogenous inhibitors of nitric oxide synthesis, have been associated with increased cardiovascular risk, the role that ADMA and SDMA play in the initiation of hyperhomocysteinemia-induced endothelial dysfunction remains still controversial. In the present study, we studied the changes of circulating ADMA and SDMA in a rat model of acutely hyperhomocysteinemia-induced endothelial dysfunction. In healthy rats, endothelium-related vascular reactivity (measured as acetylcholine-induced transient decrease in mean arterial blood pressure), plasma ADMA and SDMA, total plasma homocysteine (tHcy), cysteine and glutathione were measured before and 2, 4 and 6 h after methionine loading or vehicle. mRNA expression of hepatic dimethylarginine dimethylaminohydrolase-1 (DDAH1), a key protein responsible for ADMA metabolism, was measured 6 h after the methionine loading or the vehicle. Expectedly, methionine load induced a sustained increase in tHcy (up to 54.9 ± 1.9 µM) and a 30 % decrease in vascular reactivity compared to the baseline values. Plasma ADMA and SDMA decreased transiently after the methionine load. Hepatic mRNA expression of DDAH1, cathepsin D, and ubiquitin were significantly lower 6 h after the methionine load than after the vehicle. The absence of an elevation of circulating ADMA and SDMA in this model suggests that endothelial dysfunction induced by acute hyperhomocysteinemia cannot be explained by an up-regulation of protein arginine methyltransferases or a down-regulation of DDAH1. In experimental endothelial dysfunction induced by acute hyperhomocysteinemia, down-regulation of the proteasome is likely to dampen the release of ADMA and SDMA in the circulation.

Dietary polyunsaturated fatty acids increase survival and decrease bacterial load during septic Staphylococcus aureus infection and improve neutrophil function in mice.

Severe infection, including sepsis, is an increasing clinical problem that causes prolonged morbidity and substantial mortality. At present, antibiotics are essentially the only pharmacological treatment for sepsis. The incidence of resistance to antibiotics is increasing; therefore, it is critical to find new therapies for sepsis. Staphylococcus aureus is a major cause of septic mortality. Neutrophils play an important role in the defense against bacterial infections. We have shown that a diet with high levels of dietary saturated fatty acids decreases survival in septic mice, but the mechanisms behind this remain elusive. The aim of the present study was to investigate how the differences in dietary fat composition affect survival and bacterial load after experimental septic infection and neutrophil function in uninfected mice. We found that, after S. aureus infection, mice fed a polyunsaturated high-fat diet (HFD-P) for 8 weeks had increased survival and decreased bacterial load during sepsis compared with mice fed a saturated high-fat diet (HFD-S), similar to mice fed a low-fat diet (LFD). Uninfected mice fed HFD-P had a higher frequency of neutrophils in bone marrow than mice fed HFD-S. In addition, mice fed HFD-P had a higher frequency of neutrophils recruited to the site of inflammation in response to peritoneal injection of thioglycolate than mice fed HFD-S. Differences between the proportion of dietary protein and carbohydrate did not affect septic survival at all. In conclusion, polyunsaturated dietary fat increased both survival and efficiency of bacterial clearance during septic S. aureus infection. Moreover, this diet increased the frequency and chemotaxis of neutrophils, key components of the immune response to S. aureus infections.

Important role of autophagy in endothelial cell response to ionizing radiation.

OBJECTIVES: Vasculature damage is an important contributor to the side-effects of radiotherapy. The aim of this study is to provide insights into the radiobiology of the autophagic response of endothelial cells. METHODS AND MATERIALS: Human umbilical vascular endothelial cells (HUVEC) were exposed to 2 Gy of ionizing radiation (IR) and studied using confocal microscopy and western blot analysis, at 4 and 8 days post-irradiation. The role of autophagy flux in HUVEC radio-sensitivity was also examined. RESULTS: IR-induced accumulation of LC3A(+), LC3B(+) and p62 cytoplasmic vacuoles, while in double immunostaining with lysosomal markers (LAMP2a and CathepsinD) repression of the autophagolysosomal flux was evident. Autophagy-related proteins (ATF4, HIF1α., HIF2α, Beclin1) were, however, induced excluding an eventual repressive effect of radiation on autophagy initiating protein expression. Exposure of HUVEC to SMER28, an mTOR-independent inducer of autophagy, enhanced proLC3 and LC3A, B-I protein expression and accelerated the autophagic flux. Pre-treatment of HUVEC with SMER28 protected against the blockage of autophagic flux induced by IR and conferred radio-resistance. Suppression of LC3A/LC3B proteins with siRNAs resulted in radio-sensitization. CONCLUSIONS: The current data provide a rationale for the development of novel radioprotection policies targeting the autophagic pathway.

Vasoinhibins, N-terminal mouse prolactin fragments, participate in mammary gland involution.

Vasoinhibins are a family of peptides that act on endothelial cells to suppress angiogenesis and promote apoptosis-mediated vascular regression. Vasoinhibins include the N-terminal fragments from prolactin (PRL), GH, and placental lactogen. One of the vasoinhibins, the N-terminal PRL fragment of 16 kDa, is generated by the lysosomal representative protease cathepsin D (Cath D). Because the normal growth and involution of the mammary gland (MG) are profoundly affected by the expansion and regression of blood vessels and also because PRL stimulates the growth and differentiation of MG, we proposed that intact PRL produced during lactation contributes to MG angiogenesis and increased blood flow, whereas during involution, the N-terminal PRL fragment would have proapoptotic effects on mammary epithelial cells (MECs). Therefore, we investigated the production of the N-terminal PRL fragment and its direct effect on the MG. Mouse PRL (mPRL) was proteolytically cleaved by Cath D between amino acids 148 and 149. N-terminal PRL fragment and Cath D expression increased during MG involution. Furthermore, incubation of MG fragments and MCF7 with recombinant 16 kDa mPRL revealed a proapoptotic effect in MECs. Ectopic mPRL in MECs was cleaved to 16 kDa PRL by Cath D in the MG lysosomal fraction. The majority of PRL derived from pituitary gland was cleaved to 16 kDa PRL in culture medium. Therefore, N-terminal PRL fragment increases during the involution period, has a proapoptotic effect on MECs, and is mainly generated by secreted Cath D in the extracellular space of MG.

Identification and functional analysis of the cathepsin D gene promoter of Bombyx mori.

The gene encoding cathepsin D of silkworm, Bombyx mori (BmCatD) is specifically expressed in the larval fat body and pupal gut, and plays an important role in the programmed cell death during metamorphosis. To identify element involved in this transcription-dependent spatial restriction, truncation and deletion of the 5' terminal from the BmCatD promoter were conducted in vivo. The recombinant AcMNPV vector (Autographa californica multiple nucleopolyhedrovirus) with a dual-luciferase quantitative assay system was used as the transfer. A 289 bp DNA sequence (-1,214 to -925) upstream of the transcriptional start site is found to be responsible for promoting tissue-specific transcription. Further analysis of a series of deletion within the 289 bp region of overlapping deletion showed that a 33 bp region (-1,071 to -1,038) sequence suppresses the ectopic expression of the BmCatD promoter. These results suggest that this 33 bp region could function as a promoter element in the tissue-specificity expression.

Significance of the tumor protease cathepsin D for the biology of breast cancer.

Cathepsin D is a protease involved in the metastasis and angiogenesis of mammary carcinomas. This review analyzes the significance of the tumor protease cathepsin D in mammary carcinomas as a tumor marker. We present a systematic overview based on a selective Medline search. Cathepsin D is expressed in mammary carcinomas and exhibits higher expression in invasive ductal carcinomas compared with lobular carcinomas. Nodal positive carcinomas showed reduced cathepsin D expression compared to lymph node metastases, and increased expression has been observed in hormone-receptor negative tumors. Thus, the expression of cathepsin varies between the two histological types. Increased cathepsin-D expression in acinar affection has also been described. The lack of an association of cathepsin D with known prognostic factors such as CA15-3, ERalpha and ERbeta does not prevent it from being using as a tumor marker. Cathepsin has already been used along with other genes as a prognostic parameter for carcinoma patients in gene arrays.

Knockdown of cathepsin D in zebrafish fertilized eggs determines congenital myopathy.

CD (cathepsin D) is a ubiquitous lysosomal hydrolase involved in a variety of pathophysiological functions, including protein turnover, activation of pro-hormones, cell death and embryo development. CD-mediated proteolysis plays a pivotal role in tissue and organ homoeostasis. Altered expression and compartmentalization of CD have been observed in diseased muscle fibres. Whether CD is actively involved in muscle development, homoeostasis and dystrophy remains to be demonstrated. Zebrafish (Danio rerio) is emerging as a valuable 'in vivo' vertebrate model for muscular degeneration and congenital myopathies. In this work, we report on the perturbance of the somitic musculature development in zebrafish larvae caused by MPO (morpholino)-mediated silencing of CD in oocytes at the time of fertilization. Restoring CD expression, using an MPO-non-matching mutated mRNA, partially rescued the normal phenotype, confirming the indispensable role of CD in the correct development and integrity of the somitic musculature. This is the first report showing a congenital myopathy caused by CD deficiency in a vertebrate experimental animal model.

Expression of cathepsins S and D signals a distinctive biochemical trait in CD34+ hematopoietic stem cells of relapsing-remitting multiple sclerosis patients.

BACKGROUND: The elucidation of mechanistic aspects of relapsing-remitting multiple sclerosis (RRMS) pathogenesis may offer valuable insights into diagnostic decisions and medical treatment. RESULTS: Two lysosomal proteases, cathepsins S and D (CatS and CatD), display an exclusive pattern of expression in CD34(+) hematopoietic stem cells (HSCs) from peripheral blood of acute MS (A-MS) patients (n = 20). While both enzymes normally exist as precursor forms in the HSCs of healthy individuals (n = 30), the same cells from A-MS patients consistently exhibit mature enzymes. Further, mature cathepsins are expressed at lower rates in stable MS subjects (S-MS, n = 15) and revert to precursor proteins after interferon-ß1a treatment (n = 5). Mature CatD and CatS were induced in HSCs of healthy donors that were either co-cultured with PBMCs of A-MS patients or exposed to their plasma, suggesting a functional involvement of soluble agents. Following HSC exposure to several cytokines known to be implicated in MS, and based on relative cytokine levels displayed in A-MS, S-MS and control individuals, we identified IL-16 as a specific cell signaling factor associated with cathepsin processing. CONCLUSIONS: These data point to an evident correlation between CatS and CatD expression and MS clinical stage, and define a biochemical trait in HSCs with functional, medical, and diagnostic relevance.

Cathepsin B, D, and L regulation in cyclosporin A-mediated gingival hyperplasia of a patient with sarcoidosis.

BACKGROUND: Cyclosporin A (CsA) is an immunosuppressant with side effects including gingival hyperplasia. Sarcoidosis is a systemic disease characterized by granulomas. Here, we report on a rare case of sarcoidosis with gingival hyperplasia to clarify whether clinical observation corresponds to in vitro results. METHODS: Gingival fibroblasts (HGFs) were isolated from healthy gingiva and cultured with CsA. Total RNA was collected and expression of mRNAs examined using semi-quantitative RT-PCR analysis. Cathepsin B, D, and L expression in overgrown gingiva of the patient was examined by immunohistochemistry. RESULTS: Cathepsin D, L, and vascular endothelial growth factor (VEGF)165 mRNA were markedly suppressed in CsA-treated HGFs, whereas cathepsin B, matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA were not reduced. Next, the decrease of cathepsin B and L expression in enlarged gingiva was observed, whereas an increase of cathepsin D expression was observed. Clinically, the enlarged gingival lesions were fully resolved by performing oral infection control. CONCLUSIONS: Cathepsins regulation might be an important factor in the development of CsA-mediated gingival hyperplasia.

Peroxisome proliferator-activated receptor γ-regulated cathepsin D is required for lipid antigen presentation by dendritic cells.

It is well established that dendritic cells (DCs) take up, process, and present lipid Ags in complex with CD1d molecules to invariant NKT cells. The lipid-activated transcription factor, peroxisome proliferator-activated receptor γ (PPARγ), has previously been shown to regulate CD1d expression in human monocyte-derived DCs, providing a link between lipid metabolism and lipid Ag presentation. We report that PPARγ regulates the expression of a lysosomal protease, cathepsin D (CatD), in human monocyte-derived DCs. Inhibition of CatD specifically reduced the expansion of invariant NKT cells and furthermore resulted in decreased maturation of saposins, a group of lipid transfer proteins required for lysosomal lipid Ag processing and loading. These results reveal a novel mechanism of lipid Ag presentation and identify CatD as a key component of this machinery and firmly place PPARγ as the transcriptional regulator linking lipid metabolism and lipid Ag processing.

Enzymatic activity of free-prostate-specific antigen (f-PSA) is not required for some of its physiological activities.

BACKGROUND: Prostate specific antigen (PSA) is a well known biomarker for early diagnosis and management of prostate cancer. Furthermore, PSA has been documented to have anti-angiogenic and anti-tumorigenic activities in both in vitro and in vivo studies. However, little is known about the molecular mechanism(s) involved in regulation of these processes, in particular the role of the serine-protease enzymatic activity of PSA. METHODS: Enzymatic activity of PSA isolated directly from seminal plasma was inhibited specifically (>95%) by incubation with zinc2+ . Human umbilical vein endothelial cells (HUVEC) were utilized to compare/contrast the physiological effects of enzymatically active versus inactive PSA. RESULTS: Equimolar concentrations of enzymatically active PSA and PSA enzymatically inactivated by incubation with Zn2+ had similar physiological effects on HUVEC, including inhibiting the gene expression of pro-angiogenic growth factors, like VEGF and bFGF, and up-regulation of expression of the anti-angiogenic growth factor IFN-γ; suppression of mRNA expression for markers of blood vessel development, like FAK, FLT, KDR, TWIST-1; P-38; inhibition of endothelial tube formation in the in vitro Matrigel Tube Formation Assay; and inhibition of endothelial cell invasion and migration properties. DISCUSSION: Our data provides compelling evidence that the transcriptional regulatory and the anti-angiogenic activities of human PSA are independent of the innate enzymatic activity.

Preclinical validation of salivary biomarkers for primary Sjögren's syndrome.

OBJECTIVE: Sjögren's syndrome (SS) is a systemic autoimmune disease with a variety of presenting symptoms that may delay its diagnosis. We previously discovered a number of candidate salivary biomarkers for primary SS using both mass spectrometry and expression microarray analysis. In the current study, we aimed to verify these candidate biomarkers in independent patient populations and to evaluate their predictive values for primary SS detection. METHODS: In total, 34 patients with primary SS, 34 patients with systemic lupus erythematosus (SLE), and 34 healthy individuals were enrolled for the validation studies. Salivary protein biomarkers were measured using either Western blotting or enzyme-linked immunosorbent assay, and the messenger RNA (mRNA) biomarkers were measured using quantitative polymerase chain reaction. Statistical analysis was performed using R software, version 2.9. RESULTS: Three protein biomarkers (cathepsin D [CPD], α-enolase, and ß2-microglobulin [ß2m]) and 3 mRNA biomarkers (myeloid cell nuclear differentiation antigen [MNDA], guanylate binding protein 2 [GBP-2], and low-affinity IIIb receptor for the Fc fragment of IgG) were significantly elevated in patients with primary SS compared with both SLE patients and healthy controls. The combination of 3 protein biomarkers, CPD, α-enolase, and ß2m, yielded a receiver operating characteristic (ROC) value of 0.99 in distinguishing primary SS from healthy controls. The combination of protein biomarkers ß2m and 2 mRNA biomarkers, MNDA and GBP-2, reached an ROC of 0.95 in discriminating primary SS from SLE. CONCLUSION: We have successfully verified a panel of protein and mRNA biomarkers that can discriminate primary SS from both SLE and healthy controls. If further validated in patients with primary SS and those with sicca symptoms but no autoimmune disease, these biomarkers may lead to a simple yet highly discriminatory clinical tool for diagnosis of primary SS.