Dynamic expression of VDR and 1-­alpha-­hydroxylase in differentiated and re-­differentiated human articular chondrocytes

Abstract of a presentation at a conference of the International Cartilage Repair Society.Purpose: The goal was to investigate potential roles played by vitamin D in the regulation of joint cartilage biology. We studied the expression of two central elements of vitamin D metabolism, namely the vitamin D receptor and its converting enzyme 1­α­hydroxylase in human knee cartilage and chondrocytes. Methods and Materials: Expression of receptor and enzyme was determined by immunohistochemistry/immunofluorescence, reversetranscriptase PCR and western blot on differentiated, de­differentiated and re­differentiated chondrocytes. Cartilage was harvested from a macroscopically healthy looking area of the lateral femoral condyle during knee replacement surgery in 4 otherwise healthy patients aged 50­70. Suspension cultures of differentiated chondrocytes were established by short enzymatic digestion of cartilage using Collagenase XI and further incubation in non­adherent vessels. De­differentiated cells were the result of serial expansion of chondrocytes during 4 weeks after isolation in monolayers cultures. Chondrocyte re­differentiation was achieved by propagating cell pellets for 3 weeks in the presence of chondro­inductive morphogens. Results: Both protein and gene expression of vitamin D receptor appear to be very low or undetectable in native cartilage and/or differentiated chondrocytes. In contrast, receptor expression was upregulated in dedifferentiated cells after monolayer expansion, however, this upregulation was lost when cells regained chondrogenic phenotype in 3D pellets. The expression of 1­α­hydroxylase was observed on the superficial layer of chondrocytes in native cartilage, which correlated with weak but detectable outcomes by PCR and western blot on differentiated cultures. Similarly, levels of the enzyme were increased after cell expansion in monolayers and decreased in 3D pellet cultures. Conclusion: Our study uncover a previously unknown regulation of vitamin D receptor between differentiated and redifferentiated phenotypes in cartilage cells. Furthermore, this study is pioneering on investigating the expression of 1­α­hydroxylase in cartilage tissue and chondrocytes. Further work is needed to ascertain if receptor and enzyme expression is regulated in disease conditions or affected by inflammatory environments

Expression and function of Leukotriene B4 receptors in human articular chondrocytes

Leukotriene B4 (LTB4) is linked to osteoarthritis (OA) development however the expression of LTB4 receptors in cartilage cells and the physiological effects of LTB4 on cartilage tissue remain unknown. In this study we find that human articular chondrocytes express LTB4 receptors and that these receptors are functional, however, LTB4 does not seem to affect importantly some primary chondrocyte functions

The role of formyl peptide receptor 1 (FPR1) in neuroblastoma tumorigenesis

Published version. Source at http://dx.doi.org/10.1186/s12885-016-2545-1 Background: Formyl peptide receptor 1 (FPR1) is a G protein-coupled receptor mainly expressed by the cells of myeloid origin, where it mediates the innate immune response to bacterial formylated peptides. High expression of FPR1 has been detected in various cancers but the function of FPR1 in tumorigenesis is poorly understood. Methods: Expression of FPR1 in neuroblastoma cell lines and primary tumors was studied using RT-PCR, western blotting, immunofluorescence and immunohistochemistry. Calcium mobilization assays and western blots with phospho-specific antibodies were used to assess the functional activity of FPR1 in neuroblastoma. The tumorigenic capacity of FPR1 was assessed by xenografting of neuroblastoma cells expressing inducible FPR1 shRNA, FPR1 cDNA or control shRNA in nude mice. Results: FPR1 is expressed in neuroblastoma primary tumors and cell lines. High expression of FPR1 corresponds with high-risk disease and poor patient survival. Stimulation of FPR1 in neuroblastoma cells using fMLP, a selective FPR1 agonist, induced intracellular calcium mobilization and activation of MAPK/Erk, PI3K/Akt and P38-MAPK signal transduction pathways that were inhibited by using Cyclosporin H, a selective receptor antagonist for FPR1. shRNA knock-down of FPR1 in neuroblastoma cells conferred a delayed xenograft tumor development in nude mice, whereas an ectopic overexpression of FPR1 promoted augmented tumorigenesis in nude mice. Conclusion: Our data demonstrate that FPR1 is involved in neuroblastoma development and could represent a therapy option for the treatment of neuroblastoma

Cortisol levels and cognitive profile in major depression: A comparison of currently and previously depressed patients

Source at https://doi.org/10.1016/j.psyneuen.2018.08.024.The association between depressive symptoms and elevated cortisol levels, and depression and cognitive functioning, has been less robust in outpatients with symptoms in the mild to moderate range. Furthermore, the association between elevated cortisol levels and cognitive functioning is unclear. In the present study, currently depressed (n = 37), previously depressed (n = 81) and never depressed controls (n = 50) were assessed on a range of neuropsychological measures. Salivary cortisol was measured in the morning and evening. Participants with current depression were non-hospitalized and had symptoms predominately in the mild to moderate range. Elevated salivary evening cortisol, but not morning cortisol, was significantly related to depressive symptoms. The difference in cortisol levels between the previously depressed group and the never depressed controls was not significant. The groups had significantly different cognitive profiles, with the currently depressed performing poorer on tasks related to working memory compared to the never depressed controls. Both the currently and previously depressed performed worse on attentional tasks. The findings indicate that outpatients with mild to moderate depression have elevated cortisol levels and limited mild cognitive impairments. Furthermore, mild impairments in attention may persist after remission, indicating that this could be a trait-marker in depression. The present study did not find support for a significant relationship between cortisol and cognitive functioning

Dynamic expression of VDR and 1-­alpha-­hydroxylase in differentiated and re-­differentiated human articular chondrocytes

Purpose: The goal was to investigate potential roles played by vitamin D in the regulation of joint cartilage biology. We studied the expression of two central elements of vitamin D metabolism, namely the vitamin D receptor and its converting enzyme 1­α­hydroxylase in human knee cartilage and chondrocytes. Methods and Materials: Expression of receptor and enzyme was determined by immunohistochemistry/immunofluorescence, reversetranscriptase PCR and western blot on differentiated, de­differentiated and re­differentiated chondrocytes. Cartilage was harvested from a macroscopically healthy looking area of the lateral femoral condyle during knee replacement surgery in 4 otherwise healthy patients aged 50­70. Suspension cultures of differentiated chondrocytes were established by short enzymatic digestion of cartilage using Collagenase XI and further incubation in non­adherent vessels. De­differentiated cells were the result of serial expansion of chondrocytes during 4 weeks after isolation in monolayers cultures. Chondrocyte re­differentiation was achieved by propagating cell pellets for 3 weeks in the presence of chondro­inductive morphogens. Results: Both protein and gene expression of vitamin D receptor appear to be very low or undetectable in native cartilage and/or differentiated chondrocytes. In contrast, receptor expression was upregulated in dedifferentiated cells after monolayer expansion, however, this upregulation was lost when cells regained chondrogenic phenotype in 3D pellets. The expression of 1­α­hydroxylase was observed on the superficial layer of chondrocytes in native cartilage, which correlated with weak but detectable outcomes by PCR and western blot on differentiated cultures. Similarly, levels of the enzyme were increased after cell expansion in monolayers and decreased in 3D pellet cultures. Conclusion: Our study uncover a previously unknown regulation of vitamin D receptor between differentiated and redifferentiated phenotypes in cartilage cells. Furthermore, this study is pioneering on investigating the expression of 1­α­hydroxylase in cartilage tissue and chondrocytes. Further work is needed to ascertain if receptor and enzyme expression is regulated in disease conditions or affected by inflammatory environments

Co-expression of 1α-hydroxylase and vitamin D receptor in human articular chondrocytes

Background: The aim was to investigate whether resident chondrocytes in human articular cartilage and in subculture express vitamin D receptor (VDR) and the enzyme that hydroxylates the prohormone 25(OH)D3 to the active hormone 1α,25(OH)2D3, namely 1α-hydroxylase (CYP27B1). Any putative effects of vitamin D on chondrocytes were also explored. Methods: Cartilage from human osteoarthritic knee joints, cultured chondrocytes and cells grown in 3D spheroids were examined for the expression of VDR and 1α-hydroxylase by PCR, Western blots and immunolabelling. Receptor engagement was judged by visualizing nuclear translocation. The effects of 25(OH)D3 and 1α,25(OH)2D3 on chondrocyte functions were assessed in proliferation-, chondrogenesis- and cartilage signature-gene expression assays. The capability of chondrocytes to hydroxylate 25(OH)D3 was determined by measuring the concentration of metabolites. Finally, a putative regulation of receptor and enzyme expression by 1α,25(OH)2D3 or interleukin (IL)-1β, was investigated by Western blot. Results: Gene expression was positive for VDR in freshly isolated cells from native cartilage, cells subcultured in monolayers and in spheroids, whereas protein expression, otherwise judged low, was apparent in monolayers. Nuclear translocation of VDR occurred upon 1α,25(OH)2D3 treatment. Transcripts for 1α-hydroxylase were detected in freshly isolated cells, cultured cells and spheroids. Western blots and immunolabelling detected 1α-hydroxylase protein in all materials, while staining of tissue appeared confined to cells at the superficial layer. A dose-dependent 1α,25(OH)2D3 production was measured when the enzyme substrate was supplied to cell cultures. Western blots revealed that the VDR, but not 1α-hydroxylase, was induced by IL-1β treatment in adherent cells. Proliferation in monolayers was enhanced by both 25(OH)D3 and 1α,25(OH) 2D3, and both compounds had negative effects on chondrogenesis and cartilage-matrix genes. Conclusions: VDR expression in resident cartilage chondrocytes, generally considered differentiated cells, is elusive. A similar pattern applies for redifferentiated chondrocytes in spheroid cultures, whereas dedifferentiated cells, established in monolayers, stably express VDR. Both 25(OH)D3 and 1α,25(OH)2D3 are able to potentiate cell proliferation but have a negative impact in proteoglycan synthesis. Chondrocytes express 1α-hydroxylase and may contribute to the production of 1α,25(OH)2D3 into the joint environment. Effects of vitamin D could be unfavourable in the context of cartilage matrix synthesis

Human articular chondrocytes express functional Leukotriene B4 receptors

Leukotriene B4 (LTB4) is a potent chemoattractant associated with the development of osteoarthritis (OA), while its receptors BLT1 and BLT2 have been found in synovium and subchondral bone. In this study, we have investigated whether these receptors are also expressed by human cartilage cells and their potential effects on cartilage cells. The expression of LTB4 receptors in native tissue and cultured cells was assessed by immunohistochemistry, immunocytochemistry, polymerase chain reaction (PCR) and electron microscopy. The functional significance of the LTB4 receptor expression was studied by Western blotting, using phospho-specific antibodies in the presence or absence of receptor antagonists. In further studies, the secretion of pro-inflammatory cytokines, growth factors and metalloproteinases by LTB4-stimulated chondrocytes was measured by multiplex protein assays. The effects of LTB4 in cartilage signature gene expression in cultured cells were assessed by quantitative PCR, whereas the LTB4-promoted matrix synthesis was determined using 3D pellet cultures. Both receptors were present in cultured chondrocytes, as was confirmed by immunolabelling and PCR. The relative quantification by PCR demonstrated a higher expression of the receptors in cells from healthy joints compared with OA cases. The stimulation of cultured chondrocytes with LTB4 resulted in a phosphorylation of downstream transcription factor Erk 1/2, which was reduced after blocking BLT1 signalling. No alteration in the secretion of cytokine and metalloproteinases was recorded after challenging cultured cells with LTB4; likewise, cartilage matrix gene expression and 3D tissue synthesis were unaffected. Chondrocytes express BLT1 and BLT2 receptors, and LTB4 activates the downstream Erk 1/2 pathway by engaging the high-affinity receptor BLT1. However, any putative role in cartilage biology could not be revealed, and remains to be clarified

Effects of Age and Sex on Estimated Diabetes Prevalence Using Different Diagnostic Criteria: The Tromso OGTT Study

Hb 6.5% has recently been recommended as an alternative diagnostic criterion for diabetes. The aims of the study were to evaluate the effects of age, sex, and other factors on prevalence of diabetes and to compare risk profiles of subjects with diabetes when defined by Hb and glucose criteria. Subjects were recruited among participants in the longitudinal population-based Tromsø Study. Hb , fasting plasma glucose, and 2-hour plasma glucose were measured in 3,476 subjects. In total, 294 subjects met one or more of the diagnostic criteria for diabetes; 95 met the Hb criterion only, 130 met the glucose criteria only, and 69 met both. Among subjects with diabetes detected by glucose criteria (regardless of Hb ), isolated raised 2-hour plasma glucose was more common in subjects aged ≥ 60 years as compared to younger subjects and in elderly women as compared to elderly men. Subjects with diabetes detected by glucose criteria only had worse cardiometabolic risk profiles than those detected by Hb only. In conclusion, the current Hb and glucose criteria defined different subjects with diabetes with only modest overlap. Among a substantial proportion of elderly subjects, and especially elderly women, the 2-hour plasma glucose was the only abnormal value

Effects of Vitamin D binding protein phenotypes and Vitamin D supplementation on serum total 25(OH)D and directly measured free 25(OH)D

Objective:To determine the relationship between serum total 25-hydroxyvitamin D (25(OH)D), directly measured free 25(OH)D and calculated free 25(OH)D with regard to vitamin D-binding protein (DBP) phenotypes, sex, BMI, age and season, and their interrelationship to vitamin D supplementation.Design, patients and interventions:A randomized controlled trial with 20 000 IU of vitamin D3 per week or placebo for 12 months was designed. A total of 472 subjects, 236 in each of the intervention groups, were included in the analyses.Main outcome measures:Baseline serum concentrations and increases in serum total 25(OH)D, directly measured free 25(OH)D, calculated free 25(OH)D and DBP.Results:Serum total 25(OH)D and DBP concentrations were significantly lower in subjects with the phenotype Gc2/Gc2 compared to phenotypes with the Gc1S allele, and lower in males compared to females. When using directly measured free 25(OH)D, the differences related to DBP phenotypes and sexes were clearly diminished. All calculated free 25(OH)D concentrations were overestimated compared to the directly measured free 25(OH)D. Serum parathyroid hormone showed an inverse correlation with all vitamin D parameters analyzed. The increases after 12 months of vitamin D supplementation were not significantly different for any of the vitamin D parameters regardless of DBP phenotype, sex or age. Supplementation with vitamin D did not affect serum DBP.Conclusion:Direct measurements of free 25(OH)D reduce the differences seen in total 25(OH)D between DBP phenotype groups and sexes, probably caused by differences in DBP concentrations. With conditions affecting serum DBP concentrations, direct measurements of free 25(OH)D should be considered