Retinoic Acid Increases Proliferation of Human Osteoclast Progenitors and Inhibits RANKL-Stimulated Osteoclast Differentiation by Suppressing RANK

It has been shown that high vitamin A intake is associated with bone fragility and fractures in both animals and humans. However, the mechanism by which vitamin A affects bones is unclear. In the present study, the direct effects of retinoic acid (RA) on human and murine osteoclastogenesis were evaluated using cultured peripheral blood CD14+ monocytes and RAW264.7 cells. Both the activity of the osteoclast marker tartrate resistant acid phosphatase (TRAP) in culture supernatant and the expression of the genes involved in osteoclast differentiation together with bone resorption were measured. To our knowledge, this is the first time that the effects of RA on human osteoclast progenitors and mature osteoclasts have been studied in vitro. RA stimulated proliferation of osteoclast progenitors both from humans and mice. In contrast, RA inhibited differentiation of the receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis of human and murine osteoclast progenitors via retinoic acid receptors (RARs). We also show that the mRNA levels of receptor activator of nuclear factor κB (RANK), the key initiating factor and osteoclast associated receptor for RANKL, were potently suppressed by RA in osteoclast progenitors. More importantly, RA abolished the RANK protein in osteoclast progenitors. This inhibition could be partially reversed by a RAR pan-antagonist. Furthermore, RA treatment suppressed the expression of the transcription factor nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and increased the expression of interferon regulatory factor-8 (IRF-8) in osteoclast progenitors via RARs. Also, RA demonstrated differential effects depending on the material supporting the cell culture. RA did not affect TRAP activity in the culture supernatant in the bone slice culture system, but inhibited the release of TRAP activity if cells were cultured on plastic. In conclusion, our results suggest that retinoic acid increases proliferation of human osteoclast progenitors and that it inhibits RANK-stimulated osteoclast differentiation by suppressing RANK

Connective tissue growth factor in tumor pathogenesis

Key roles for connective tissue growth factor (CTGF/CCN2) are demonstrated in the wound repair process where it promotes myofibroblast differentiation and angiogenesis. Similar mechanisms are active in tumor-reactive stroma where CTGF is expressed. Other potential roles include prevention of hypoxia-induced apoptosis and promoting epithelial-mesenchymal transistion (EMT). CTGF expression in tumors has been associated to both tumor suppression and progression. For example, CTGF expression in acute lymphoblastic leukemia, breast, pancreas and gastric cancer correlates to worse prognosis whereas the opposite is true for colorectal, lung and ovarian cancer. This discrepancy is not yet understood

Regulation of hyaluronan biosynthesis : Expression in vitro and importance for tumor progression

Hyaluronan, a component of the extracellular matrix, is synthesized by either of three hyaluronan-synthesizing enzymes termed Has1, Has2 and Has3. The expression level of each Has gene varies between cell types of mesenchymal origin and is differentially regulated in response to external stimuli. For example, stimulation of mesothelial cells with PDGF-BB induced an up-regulation of the Has2 gene, whereas the Has1 and Has3 genes remained unaffected. The induction of Has2 gene expression correlated well with increased Has2 protein levels and accumulation of hyaluronan. Moreover, treatment of mesothelial cells with hydrocortisone suppressed hyaluronan synthesis in cell culture primarily through down-regulation of the Has2 gene. Thus, among the Has isoforms, Has2 seems to be most markedly regulated in response to external stimuli. In an attempt to investigate the importance of hyaluronan in tumor progression, the hyaluronan synthesizing enzyme Has2 and the hyaluronan degrading enzyme Hyal1 were over-expressed in a rat colon adenocarcinoma cell line, PROb. We found that Has2 gene over-expression in colon carcinoma cells promoted cell growth in vitro and progression of transplantable tumors. In contrast, over-expression of Hyal1 lead to a considerable reduction of growth rates both in vivo and in vitro. A linear correlation between tumor growth rate and hyaluronan amount in tumor tissue was observed. In another tumor model, experimental anaplastic thyroid carcinoma, the effects of TGF-β inhibition on hyaluronan and collagen contents in tumor xenografts were investigated. We found that inhibition of TGF-β, a stimulator of hyaluronan and collagen synthesis, lead to reduced collagen deposition whereas the hyaluronan levels in stromal tissue only marginally differed. Our results indicate that a high ratio of collagen to hyaluronan may be characteristic of a pathogenic mechanism that leads to elevated interstitual tumor pressure

Regulation of hyaluronan biosynthesis : Expression in vitro and importance for tumor progression

Hyaluronan, a component of the extracellular matrix, is synthesized by either of three hyaluronan-synthesizing enzymes termed Has1, Has2 and Has3. The expression level of each Has gene varies between cell types of mesenchymal origin and is differentially regulated in response to external stimuli. For example, stimulation of mesothelial cells with PDGF-BB induced an up-regulation of the Has2 gene, whereas the Has1 and Has3 genes remained unaffected. The induction of Has2 gene expression correlated well with increased Has2 protein levels and accumulation of hyaluronan. Moreover, treatment of mesothelial cells with hydrocortisone suppressed hyaluronan synthesis in cell culture primarily through down-regulation of the Has2 gene. Thus, among the Has isoforms, Has2 seems to be most markedly regulated in response to external stimuli. In an attempt to investigate the importance of hyaluronan in tumor progression, the hyaluronan synthesizing enzyme Has2 and the hyaluronan degrading enzyme Hyal1 were over-expressed in a rat colon adenocarcinoma cell line, PROb. We found that Has2 gene over-expression in colon carcinoma cells promoted cell growth in vitro and progression of transplantable tumors. In contrast, over-expression of Hyal1 lead to a considerable reduction of growth rates both in vivo and in vitro. A linear correlation between tumor growth rate and hyaluronan amount in tumor tissue was observed. In another tumor model, experimental anaplastic thyroid carcinoma, the effects of TGF-β inhibition on hyaluronan and collagen contents in tumor xenografts were investigated. We found that inhibition of TGF-β, a stimulator of hyaluronan and collagen synthesis, lead to reduced collagen deposition whereas the hyaluronan levels in stromal tissue only marginally differed. Our results indicate that a high ratio of collagen to hyaluronan may be characteristic of a pathogenic mechanism that leads to elevated interstitual tumor pressure

Daytime melatonin levels in saliva are associated with inflammatory markers and anxiety disorders

BACKGROUND: The bidirectional interaction between melatonin and the immune system has largely gone unexplored in a clinical context and especially in a psychiatric population. This study explored the association between melatonin during the day and inflammatory cytokines in young adult patients seeking psychiatric care. METHODS: Samples and data were collected from 108 young adults (mean age 21, SD = 2) at an outpatient clinic for affective disorders. Daytime saliva melatonin levels were analyzed with enzyme-linked immunosorbent assay (ELISA) in relation to normalized serum expression levels of 72 inflammatory markers in a proximity extension assay (PEA). In a post hoc analysis the markers associated with melatonin were tested in a generalized linear model to see whether there is a relationship to anxiety disorder or depression. RESULTS: After Bonferroni correction for multiple testing, melatonin levels at 11:00 were positively correlated with CD5 (p = 4.2e-4). Melatonin levels after lunch were correlated with CCL2/MCP-1 (p = 4.2e-4), CCL3/MPI-1α (p = 6.5e-4) and VEGF-A (p = 5.3e-6). In the generalized linear model, positive associations were found for the presence of any anxiety disorder with melatonin after lunch (p = 0.046), VEGF-A (p = 0.001) and CCL3/MPI-1α (p = 0.001). CONCLUSION: Daytime saliva levels of melatonin were related to several inflammatory markers in young adults with psychiatric disorders. This observation likely reflects the bidirectional relationship between melatonin production and the immune system. These findings may have relevance for the understanding of psychiatric disorders and other conditions associated with low-grade inflammation.Authors and Title in Thesis List of papers:I. Sundberg, A. Jacobson, M. Ramklint, L. Ekselius, and J. Cunningham “Daytime Melatonin Levels in Saliva are Associated with Inflammatory Markers and Anxiety Disorders in Young Adults with Psychiatric Disease”</p

The Free Hormone Hypothesis : Is Free Serum 25-Hydroxyvitamin D a Better Marker for Bone Mineral Density in Older Women?

It is presently unclear whether free serum 25-hydroxyvitamin D (S-25(OH)D) better reflects bone health than total S-25(OH)D. We have previously shown that summer total S-25(OH)D values are more useful to predict bone mineral density (BMD) than winter values. Our objective was therefore to compare the relative importance of free and total S-25(OH)D for BMD by season. BMD was measured by dual-energy X-ray absorptiometry (DXA) in 5002 Swedish women (mean age 68 years) randomly selected from a large population-based longitudinal cohort study. Free S-25(OH)D was analyzed by a commercial ELISA and total S-25(OH)D by HPLC-tandem mass spectrometry (MS/MS). Free and total S-25(OH)D co-varied with season, with 26% and 29% higher values in August compared with those in January-March (nadir). There were no differences in mean BMD between categories of free or total S-25(OH)D in samples collected during winter. Women with higher total S-25(OH)D measured during summer had higher BMD at the total hip. Compared with women who had total S-25(OH)D values above 80 nmol/L during summer, adjusted BMD at the total hip was 6% (95% CI, 1% to 11%) lower for S-25(OH)D concentrations between 30 and 40 mmol/L, and 11% (95% CI, 3% to 19%) lower for those with total S-25(OH)D &lt;30 nmol/L. In contrast, free S-25(OH)D measured during summer was not associated with BMD. Compared with women who had highest free S-25(OH)D measured during summer (&gt;8.8 pmol/L), those with intermediate (2.4-3.5 pmol/L) and lowest (&lt;2.4 pmol/L) free S-25(OH)D during summer did not have lower total hip BMD values (3% [95% CI, -2% to 7%] and -2% [95% CI, -8% to 4%]). In addition, we found no added value for the prediction of BMD with the combined measurement of total and free S-25(OH)D during summer or winter. We conclude that vitamin D status assessed by direct measurements of free S-25(OH)D does not reflect BMD better than total S-25(OH)D. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research

Vitamin A Is a Negative Regulator of Osteoblast Mineralization

An excessive intake of vitamin A has been associated with an increased risk of fractures in humans. In animals, a high vitamin A intake leads to a reduction of long bone diameter and spontaneous fractures. Studies in rodents indicate that the bone thinning is due to increased periosteal bone resorption and reduced radial growth. Whether the latter is a consequence of direct effects on bone or indirect effects on appetite and general growth is unknown. In this study we therefore used pair-feeding and dynamic histomorphometry to investigate the direct effect of a high intake of vitamin A on bone formation in rats. Although there were no differences in body weight or femur length compared to controls, there was an approximately halved bone formation and mineral apposition rate at the femur diaphysis of rats fed vitamin A. To try to clarify the mechanism(s) behind this reduction, we treated primary human osteoblasts and a murine preosteoblastic cell line (MC3T3-E1) with the active metabolite of vitamin A; retinoic acid (RA), a retinoic acid receptor (RAR) antagonist (AGN194310), and a Cyp26 inhibitor (R115866) which blocks endogenous RA catabolism. We found that RA, via RARs, suppressed in vitro mineralization. This was independent of a negative effect on osteoblast proliferation. Alkaline phosphatase and bone gamma carboxyglutamate protein (Bglap, Osteocalcin) were drastically reduced in RA treated cells and RA also reduced the protein levels of Runx2 and Osterix, key transcription factors for progression to a mature osteoblast. Normal osteoblast differentiation involved up regulation of Cyp26b1, the major enzyme responsible for RA degradation, suggesting that a drop in RA signaling is required for osteogenesis analogous to what has been found for chondrogenesis. In addition, RA decreased Phex, an osteoblast/osteocyte protein necessary for mineralization. Taken together, our data indicate that vitamin A is a negative regulator of osteoblast mineralization