Mass spectrometry-based proteomics identify novel serum osteoarthritis biomarkers.

BACKGROUND: Osteoarthritis (OA) is a slowly developing and debilitating disease, and there are no validated specific biomarkers for its early detection. To improve therapeutic approaches, identification of specific molecules/biomarkers enabling early determination of this disease is needed. This study aimed at identifying, with the use of proteomics/mass spectrometry, novel OA-specific serum biomarkers. As obesity is a major risk factor for OA, we discriminated obesity-regulated proteins to target only OA-specific proteins as biomarkers. METHODS: Serum from the Osteoarthritis Initiative cohort was used and divided into 3 groups: controls (n=8), OA-obese (n=10) and OA-non-obese (n=10). Proteins were identified and quantified from the liquid chromatography-tandem mass spectrometry analyses using MaxQuant software. Statistical analysis used the Limma test followed by the Benjamini-Hochberg method. To compare the proteomic profiles, the multivariate unsupervised principal component analysis (PCA) followed by the pairwise comparison was used. To select the most predictive/discriminative features, the supervised linear classification model sparse partial least squares regression discriminant analysis (sPLS-DA) was employed. Validation of three differential proteins was performed with protein-specific assays using plasma from a cohort derived from the Newfoundland Osteoarthritis. RESULTS: In total, 509 proteins were identified, and 279 proteins were quantified. PCA-pairwise differential comparisons between the 3 groups revealed that 8 proteins were differentially regulated between the OA-obese and/or OA-non-obese with controls. Further experiments using the sPLS-DA revealed two components discriminating OA from controls (component 1, 9 proteins), and OA-obese from OA-non-obese (component 2, 23 proteins). Proteins from component 2 were considered related to obesity. In component 1, compared to controls, 7 proteins were significantly upregulated by both OA groups and 2 by the OA-obese. Among upregulated proteins from both OA groups, some of them alone would not be a suitable choice as specific OA biomarkers due to their rather non-specific role or their strong link to other pathological conditions. Altogether, data revealed that the protein CRTAC1 appears to be a strong OA biomarker candidate. Other potential new biomarker candidates are the proteins FBN1, VDBP, and possibly SERPINF1. Validation experiments revealed statistical differences between controls and OA for FBN1 (p=0.044) and VDPB (p=0.022), and a trend for SERPINF1 (p=0.064). CONCLUSION: Our study suggests that 4 proteins, CRTAC1, FBN1, VDBP, and possibly SERPINF1, warrant further investigation as potential new biomarker candidates for the whole OA population.

14-3-3ζ Constrains insulin secretion by regulating mitochondrial function in pancreatic ß cells.

While critical for neurotransmitter synthesis, 14-3-3 proteins are often assumed to have redundant functions due to their ubiquitous expression, but despite this assumption, various 14-3-3 isoforms have been implicated in regulating metabolism. We previously reported contributions of 14-3-3ζ in ß cell function, but these studies were performed in tumor-derived MIN6 cells and systemic KO mice. To further characterize the regulatory roles of 14-3-3ζ in ß cell function, we generated ß cell-specific 14-3-3ζ-KO mice. Although no effects on ß cell mass were detected, potentiated glucose-stimulated insulin secretion (GSIS), mitochondrial function, and ATP synthesis were observed. Deletion of 14-3-3ζ also altered the ß cell transcriptome, as genes associated with mitochondrial respiration and oxidative phosphorylation were upregulated. Acute 14-3-3 protein inhibition in mouse and human islets recapitulated the enhancements in GSIS and mitochondrial function, suggesting that 14-3-3ζ is the critical isoform in ß cells. In dysfunctional db/db islets and human islets from type 2 diabetic donors, expression of Ywhaz/YWHAZ, the gene encoding 14-3-3ζ, was inversely associated with insulin secretion, and pan-14-3-3 protein inhibition led to enhanced GSIS and mitochondrial function. Taken together, this study demonstrates important regulatory functions of 14-3-3ζ in the regulation of ß cell function and provides a deeper understanding of how insulin secretion is controlled in ß cells.

L-PGDS deficiency accelerated the development of naturally occurring age-related osteoarthritis.

Osteoarthritis (OA) is the most common musculoskeletal disorder among the elderly. It is characterized by progressive cartilage degradation, synovial inflammation, subchondral bone remodeling and pain. Lipocalin prostaglandin D synthase (L-PGDS) is responsible for the biosynthesis of PGD2, which has been implicated in the regulation of inflammation and cartilage biology. This study aimed to evaluate the effect of L-PGDS deficiency on the development of naturally occurring age-related OA in mice. OA-like structural changes were assessed by histology, immunohistochemistry, and micro-computed tomography. Pain related behaviours were assessed using the von Frey and the open-field assays. L-PGDS deletion promoted cartilage degradation during aging, which was associated with enhanced expression of extracellular matrix degrading enzymes, matrix metalloprotease 13 (MMP-13) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5), and their breakdown products, C1,2C, VDIPEN and NITEG. Moreover, L-PGDS deletion enhanced subchondral bone changes, but had no effect on its angiogenesis. Additionally, L-PGDS deletion increased mechanical sensitivity and reduced spontaneous locomotor activity. Finally, we showed that the expression of L-PGDS was elevated in aged mice. Together, these findings indicate an important role for L-PGDS in naturally occurring age-related OA. They also suggest that L-PGDS may constitute a new efficient therapeutic target in OA.

Role of Lipocalin-Type Prostaglandin D Synthase in Experimental Osteoarthritis.

OBJECTIVE: Lipocalin-type prostaglandin D synthase (L-PGDS) catalyzes the formation of prostaglandin D2 (PGD2 ), which has important roles in inflammation and cartilage metabolism. We undertook this study to investigate the role of L-PGDS in the pathogenesis of osteoarthritis (OA) using an experimental mouse model. METHODS: Experimental OA was induced in wild-type (WT) and L-PGDS-deficient (L-PGDS-/- ) mice (n = 10 per genotype) by destabilization of the medial meniscus (DMM). Cartilage degradation was evaluated by histology. The expression of matrix metalloproteinase 13 (MMP-13) and ADAMTS-5 was assessed by immunohistochemistry. Bone changes were determined by micro-computed tomography. Cartilage explants from L-PGDS-/- and WT mice (n = 6 per genotype) were treated with interleukin-1α (IL-1α) ex vivo in order to evaluate proteoglycan degradation. Moreover, the effect of intraarticular injection of a recombinant adeno-associated virus type 2/5 (rAAV2/5) encoding L-PGDS on OA progression was evaluated in WT mice (n = 9 per group). RESULTS: Compared to WT mice, L-PGDS-/- mice had exacerbated cartilage degradation and enhanced expression of MMP-13 and ADAMTS-5 (P < 0.05). Furthermore, L-PGDS-/- mice displayed increased synovitis and subchondral bone changes (P < 0.05). Cartilage explants from L-PGDS-/- mice showed enhanced proteoglycan degradation following treatment with IL-1α (P < 0.05). Intraarticular injection of rAAV2/5 encoding L-PGDS attenuated the severity of DMM-induced OA-like changes in WT mice (P < 0.05). The L-PGDS level was increased in OA tissues of WT mice (P < 0.05). CONCLUSION: Collectively, these findings suggest a protective role of L-PGDS in OA, and therefore enhancing levels of L-PGDS may constitute a promising therapeutic strategy.

In vivo protective effect of adipsin-deficiency on spontaneous knee osteoarthritis in aging mice.

The adipokine adipsin is an emerging mediator of human osteoarthritis (OA) progression. Here, we investigated its in vivo role in the development of spontaneous OA in aging mice. We compared articular knee joint morphology, histology in knee cartilage, synovial membrane, subchondral bone, meniscus, and anterior cruciate ligament (ACL); and chondrogenesis in the ACL from adipsin-deficient (Df-/-) and wild-type (Df+/+) 20-week- and 20-month-old mice. Serum levels of a panel of adipokines, inflammatory factors, and metalloproteases known to be implicated in OA were investigated. Data first revealed that the early manifestation of OA appeared in the ACL of 20-week-old mice, progressing to severe alterations in the 20 month-old wild-type mice. Further results demonstrated that adipsin-deficiency protected the articular tissues from spontaneous OA progression and triggered significantly higher serum levels of the adipokines adiponectin and FGF-21 while lowering levels of the inflammatory factor interleukin 6 (IL-6) in both young and old mice. This work further underlines the clinical relevance of adipsin as a novel therapeutic approach of human OA. Moreover, this study shows the potential beneficial effect of the adipokine FGF-21 against OA, and provides support for this factor to be a new biomarker and/or target of primary OA therapeutic avenues.

High in vivo levels of adipsin lead to increased knee tissue degradation in osteoarthritis: data from humans and animal models.

Objective: This study explored the role of the adipokine adipsin in OA. Methods: Control and OA articular tissues, cells and serum were obtained from human individuals. Serum adipsin levels of human OA individuals were compared with cartilage volume loss as assessed by MRI at 48 months. Human adipsin expression was determined by PCR, its production in tissues by immunohistochemistry, and in SF and serum by a specific assay. OA was surgically induced in wild-type (Df+/+) and adipsin-deficient (Df-/-) mice, and synovial membrane and cartilage processed for histology and immunohistochemistry. Results: Adipsin levels were significantly increased in human OA serum, SF, synovial membrane and cartilage compared with controls, but the expression was similar in chondrocytes, synoviocytes and osteoblasts. Multivariate analysis demonstrated that human serum adipsin levels were significantly associated (P = 0.045) with cartilage volume loss in the lateral compartment of the knee. Destabilization of the medial meniscus-Df-/- mice showed a preservation of the OA synovial membrane and cartilage lesions (P ⩽ 0.026), the latter corroborated by the decreased production of cartilage degradation products and proteases (P ⩽ 0.047). The adipsin effect is likely due to a deficient alternative complement pathway (P ⩽ 0.036). Conclusion: In human OA, higher serum adipsin levels were associated with greater cartilage volume loss in the lateral compartment, and adipsin deficiency led to a preservation of knee structure. Importantly, we documented an association between adipsin and OA synovial membrane and cartilage degeneration through the activation of the complement pathway. This study highlights the clinical relevance of adipsin as a valuable biomarker and potential therapeutic target for OA.

Modulation of stemness in a human normal intestinal epithelial crypt cell line by activation of the WNT signaling pathway.

The small intestine consists of two histological compartments composed of the crypts and the villi. The function of the adult small intestinal epithelium is mediated by four different types of mature cells: enterocytes, goblet, enteroendocrine and Paneth. Undifferentiated cells reside in the crypts and produce these four types of mature cells. The niche-related Wnt and Bmp signaling pathways have been suggested to be involved in the regulation and maintenance of the stem cell microenvironment. In our laboratory, we isolated the first normal human intestinal epithelial crypt (HIEC) cell model from the human fetal intestine and in this study we investigated the expression of a panel of intestinal stem cell markers in HIEC cells under normal culture parameters as well as under conditions that mimic the stem cell microenvironment. The results showed that short term stimulation of HIEC cells with R-spondin 1 and Wnt-3a±SB-216763, a glycogen synthase kinase 3ß (GSK3ß) inhibitor, induced ß-catenin/TCF activity and expression of the WNT target genes, cyclin D2 and LGR5. Treatment of HIEC cells with noggin, an antagonist of BMP signaling, abolished SMAD2/5/8 phosphorylation. Inducing a switch from inactive WNT/active BMP toward active WNT/inactive BMP pathways was sufficient to trigger a robust intestinal primordial stem-like cell signature with predominant LGR5, PHLDA1, PROM1, SMOC2 and OLFM4 expression. These findings demonstrate that even fully established cultures of intestinal cells can be prompted toward a CBC stem cell-like phenotype. This model should be useful for studying the regulation of human intestinal stem cell self-renewal and differentiation.

Cooperation between HNF-1alpha, Cdx2, and GATA-4 in initiating an enterocytic differentiation program in a normal human intestinal epithelial progenitor cell line.

In the intestinal epithelium, the Cdx, GATA, and HNF transcription factor families are responsible for the expression of differentiation markers such as sucrase-isomaltase. Although previous studies have shown that Cdx2 can induce differentiation in rat intestinal IEC-6 cells, no data are available concerning the direct implication of transcription factors on differentiation in human normal intestinal epithelial cell types. We investigated the role of Cdx2, GATA-4, and HNF-1alpha using the undifferentiated human intestinal epithelial crypt cell line HIEC. These transcription factors were tested on proliferation and expression of polarization and differentiation markers. Ectopic expression of Cdx2 or HNF-1alpha, alone or in combination, altered cell proliferation abilities through the regulation of cyclin D1 and p27 expression. HNF-1alpha and GATA-4 together induced morphological modifications of the cells toward polarization, resulting in the appearance of functional features such as microvilli. HNF-1alpha was also sufficient to induce the expression of cadherins and dipeptidylpeptidase, whereas in combination with Cdx2 it allowed the expression of the late differentiation marker sucrase-isomaltase. Large-scale analysis of gene expression confirmed the cooperative effect of these factors. Finally, although DcamKL1 and Musashi-1 expression were downregulated in differentiated HIEC, other intestinal stem cell markers, such as Bmi1, were unaffected. These observations show that, in cooperation with Cdx2, HNF-1alpha acts as a key factor on human intestinal cells to trigger the onset of their functional differentiation program whereas GATA-4 appears to promote morphological changes.

Oral treatment with a Brachystemma calycinum D don plant extract reduces disease symptoms and the development of cartilage lesions in experimental dog osteoarthritis: inhibition of protease-activated receptor 2.

OBJECTIVE: The aims of this study were to evaluate the effect of oral treatment with a whole plant extract of Brachystemma calycinum D don (BCD) on the development of osteoarthritic lesions and symptoms in the experimental dog anterior cruciate ligament (ACL) transection model and to document its mechanism of action. METHODS: Osteoarthritis was induced by sectioning the ACL of the right knee in crossbred dogs. There were two experimental groups (n=6-7 dogs/group): placebo and BCD extract (200 mg/kg per day) given orally for 8 weeks. Macroscopic and histopathological evaluation of cartilage lesions and immunohistochemical analysis of cartilage to assess levels of inducible nitric oxide synthase (iNOS), matrix metalloprotease 13 (MMP-13) and protease activated receptor 2 (PAR-2) were done. A gait analysis of dogs was performed. RESULTS: Treatment with BCD reduced the severity (depth) (p=0.04) and histopathological score (p<0.02) of osteoarthritis cartilage lesions. BCD treatment also significantly reduced the osteoarthritis chondrocyte level of key inflammatory and catabolic factors (iNOS, p=0.009 and MMP-13, p=0.003) as well as the level of PAR-2 (p=0.03). Dogs treated with BCD showed a significant improvement in peak vertical force measured at 8 weeks (p<0.05). CONCLUSIONS: Treatment with BCD extract exerts a positive effect on the prevention of cartilage lesions induced by joint instability, and improves joint function. This effect was associated with the inhibition of major catabolic and inflammatory mediators. This study is the first to demonstrate that a therapeutic intervention that can inhibit PAR-2 is associated with a disease-modifying osteoarthritis effect.

Integrin alpha6Bbeta4 inhibits colon cancer cell proliferation and c-Myc activity.

BACKGROUND: Integrins are known to be important contributors to cancer progression. We have previously shown that the integrin beta4 subunit is up-regulated in primary colon cancer. Its partner, the integrin alpha6 subunit, exists as two different mRNA splice variants, alpha6A and alpha6B, that differ in their cytoplasmic domains but evidence for distinct biological functions of these alpha6 splice variants is still lacking. METHODS: In this work, we first analyzed the expression of integrin alpha6A and alpha6B at the protein and transcript levels in normal human colonic cells as well as colorectal adenocarcinoma cells from both primary tumors and established cell lines. Then, using forced expression experiments, we investigated the effect of alpha6A and alpha6B on the regulation of cell proliferation in a colon cancer cell line. RESULTS: Using variant-specific antibodies, we observed that alpha6A and alpha6B are differentially expressed both within the normal adult colonic epithelium and between normal and diseased colonic tissues. Proliferative cells located in the lower half of the glands were found to predominantly express alpha6A, while the differentiated and quiescent colonocytes in the upper half of the glands and surface epithelium expressed alpha6B. A relative decrease of alpha6B expression was also identified in primary colon tumors and adenocarcinoma cell lines suggesting that the alpha6A/alpha6B ratios may be linked to the proliferative status of colonic cells. Additional studies in colon cancer cells showed that experimentally restoring the alpha6A/alpha6B balance in favor of alpha6B caused a decrease in cellular S-phase entry and repressed the activity of c-Myc. CONCLUSION: The findings that the alpha6Bbeta4 integrin is expressed in quiescent normal colonic cells and is significantly down-regulated in colon cancer cells relative to its alpha6Abeta4 counterpart are consistent with the anti-proliferative influence and inhibitory effect on c-Myc activity identified for this alpha6Bbeta4 integrin. Taken together, these findings point out the importance of integrin variant expression in colon cancer cell biology.

Differential expression of the integrins alpha6Abeta4 and alpha6Bbeta4 along the crypt-villus axis in the human small intestine.

The integrin alpha6 subunit exists as two different variants, termed alpha6A and alpha6B. These two variants have been shown to harbor potentially distinct biochemical properties but little is known about their cellular function. The aim of this work was to characterize the expression of the integrin alpha6A and B variants in relation to cell proliferation and differentiation in the human small intestinal epithelium. The results showed distinct expression patterns for the two variants along the crypt-villus axis. Indeed, proliferative cells of the crypt were found to predominantly express alpha6A, while differentiated enterocytes and Paneth cells expressed the alpha6B variant. A similar relationship was observed in intestinal cell models by competitive RT-PCR. Further studies in the Caco-2 cell model showed that manipulating the cellular balance of the two alpha6 variants can influence transcriptional activities related to cell proliferation but not differentiation. This suggests that differential expression of the alpha6 subunits is involved in the intestinal epithelial cell renewal process. Further studies will be needed to substantiate this hypothesis.

Cdx2 modulates proliferation in normal human intestinal epithelial crypt cells.

The homeobox gene Cdx2 is involved in the regulation of the expression of intestine specific markers such as sucrase-isomaltase and lactase-phlorizin hydrolase. Previous studies performed with immortalized or transformed intestinal cell lines have provided evidence that Cdx2 can promote morphological and functional differentiation in these experimental models. However, no data exist concerning the implication of this factor in normal human intestinal cell physiology. In the present work, we have investigated the role of Cdx2 in normal human intestinal epithelial crypt (HIEC) cells that lack this transcription factor. The establishment of HIEC cells expressing Cdx2 in an inducible manner shows that forced expression of Cdx2 significantly alters the proliferation of intestinal crypt cells and stimulates dipeptidylpeptidase IV expression but is not sufficient to trigger intestinal terminal differentiation. These observations suggest that Cdx2 requires additional factors to activate the enterocyte differentiation program in normal undifferentiated cells.

Repressed E-cadherin expression in the lower crypt of human small intestine: a cell marker of functional relevance.

In epithelia, abnormal expression of E-cadherin is related to pathologies involving a loss of cell polarization and/or differentiation. However, recent observations suggest that E-cadherin could also be repressed under physiological conditions, such as in some epithelial stem cell lineages. In the present work, we have analyzed E-cadherin expression in human intestinal epithelial cell progenitors and investigated its potential role. E-cadherin expression was analyzed along the crypt-villus axis by immunofluorescence on cryosections of small intestine. E-cadherin was found to be differentially expressed, being significantly weaker in the cells located at the bottom of the crypts. Surprisingly, neither the E-cadherin protein nor transcript were detected in a normal human intestinal epithelial (HIEC) crypt cell model isolated in our laboratory, whereas other E-cadherin-related components such as catenins and APC were present. Forced expression of E-cadherin in HIEC cells increased membrane-associated beta-catenin and was accompanied by the appearance of junction-like structures at the cell-cell interface. Functionally, cell kinetics and p21Cip levels were found to be altered in the E-cadherin expressing HIEC cells as compared to controls. Furthermore, a significant reduction of the migration abilities and an increase in sensitivity to anoikis were also observed. These results suggest that down-regulated expression of E-cadherin is a human intestinal crypt base cell-related feature that appears to be of functional relevance for the maintenance of the progenitor cell population.