Interleukin-18, a proinflammatory cytokine, contributes to the pathogenesis of non-thyroidal illness mainly via the central part of the hypothalamus-pituitary-thyroid axis

Objective: Proinflammatory cytokines are involved in the pathogenesis of non-thyroidal illness (NTI), its shown by studies with IL-6(-/-) and IL-12(-/-) mice. Interleukin (IL)-6 changes peripheral thyroid hormone metabolism, and IL-12 seems to be involved in the regulation of the central part of the hypothalamic-pituitary-thyroid (HPT) axis during illness. IL-18 is a proinflammatory cytokine which shares important biological properties with IL-12, such as interferon (IFN)-gamma-inducing activity. Design: By studying the changes in the HPT axis during bacterial lipopolysaccharide (LPS)-induced illness in IL-18(-/-), IFNgammaR(-/-) and wild-type (WT) mice, we wanted to unravel the putative role of IL-18 and IFNgamma in the pathogenesis of NTI. Results: LPS induced a decrease in pituitary type 1 deiodinase (D1) activity (P <0.05, ANOVA) in WT mice, but not in IL-18(-/-) mice, while the decrease in D2 activity was similar in both strains. LPS decreased serum thyroid hormone levels and liver D1 mRNA within 24h similarly in IL-18(-/-), and WT mice. The expression of IL-1, IL-6 and IFNgamma mRNA expression was sipificantly lower in IL-18(-/-) mice than in WT, while IL-12 mRNA expression was similar. IFNgammaR(-/-) mice had higher basal D1 activity in the pituitary than WT mice (P <0.05); LPS induced a decrease of D2, but not of D1, activity in the pituitary which was similar in both strains. In the liver, the LPS-induced increase in cytokine expression was not different between IFNgammaR(-/-) mice and WT mice, and the decrease in serum T-3 and T-4 levels and hepatic D1 mRNA was also similar. Conclusions: The relative decrease in serum T-3 and T-4 and liver D1 mRNA in response to LPS is similar in IL-18(-/-), IFNgammaR(-/-) and WT mice despite significant changes in hepatic cytokine induction. However, the LPS-induced decrease in D1 activity in the pituitary of WT mice is absent in IL-18(-/-) mice; in contrast, LPS did not decrease pituitary D1 activity in the IFNgammaR(-/-) mice or their WT, which might be due to the genetic background of the mice. Our results suggest that IL-18 is also involved in the regulation of the central part of the HPT axis during illnes

Probenecid as a sensitizer of bisphosphonate-mediated effects in breast cancer cells

Background: Anti-resorptive bisphosphonates (BP) are used for the treatment of osteoporosis and bone metastases. Clinical studies indicated a benefit in survival and tumor relapse in subpopulations of breast cancer patients receiving zoledronic acid, thus stimulating the debate about its anti-tumor activity. Amino-bisphosphonates in nM concentrations inhibit farnesyl pyrophosphate synthase leading to accumulation of isopentenyl pyrophosphate (IPP) and the ATP/ pyrophosphate adduct ApppI, which induces apoptosis in osteoclasts. For anti-tumor effects μM concentrations are needed and a sensitizer for bisphosphonate effects would be beneficial in clinical anti-tumor applications. We hypothesized that enhancing intracellular pyrophosphate accumulation via inhibition of probenecid-sensitive channels and transporters would sensitize tumor cells for bisphosphonates anti-tumor efficacy. Methods: MDA-MB-231, T47D and MCF-7 breast cancer cells were treated with BP (zoledronic acid, risedronate, ibandronate, alendronate) and the pyrophosphate channel inhibitors probenecid and novobiocin. We determined cell viability and caspase 3/7 activity (apoptosis), accumulation of IPP and ApppI, expression of ANKH, PANX1, ABCC1, SLC22A11, and the zoledronic acid target gene and tumor-suppressor KLF2. Results: Treatment of MDA-MB-231 with BP induced caspase 3/7 activity, with zoledronic acid being the most effective. In MCF-7 and T47D either BP markedly suppressed cell viability with only minor effects on apoptosis. Co-treatment with probenecid enhanced BP effects on cell viability, IPP/ApppI accumulation as measurable in MCF-7 and T47D cells, caspase 3/7 activity and target gene expression. Novobiocin co-treatment of MDA-MB-231 yielded identical results on viability and apoptosis compared to probenecid, rendering SLC22A family members as candidate modulators of BP effects, whereas no such evidence was found for ANKH, ABCC1 and PANX1. Conclusions: In summary, we demonstrate effects of various bisphosphonates on caspase 3/7 activity, cell viability and expression of tumor suppressor genes in breast cancer cells. Blocking probenecid- and novobiocin-sensitive channels and transporters enhances BP anti-tumor effects and renders SLC22A family members good candidates as BP modulators. Further studies will have to unravel if treatment with such BP-sensitizers translates into preclinical and clinical efficacy

Modulation of Transient Receptor Potential Channels 3 and 6 Regulates Osteoclast Function with Impact on Trabecular Bone Loss

Enhanced osteoclast formation and function is a fundamental cause of alterations to bone structure and plays an important role in several diseases impairing bone quality. Recent work revealed that TRP calcium channels 3 and 6 might play a special role in this context. By analyzing the bone phenotype of TRPC6-deficient mice we detected a regulatory effect of TRPC3 on osteoclast function. These mice exhibit a significant decrease in bone volume per tissue volume, trabecular thickness and -number together with an increased number of osteoclasts found on the surface of trabecular bone. Primary bone marrow mononuclear cells from TRPC6-deficient mice showed enhanced osteoclastic differentiation and resorptive activity. This was confirmed in vitro by using TRPC6-deficient RAW 264.7 cells. TRPC6 deficiency led to an increase of TRPC3 in osteoclasts, suggesting that TRPC3 overcompensates for the loss of TRPC6. Raised intracellular calcium levels led to enhanced NFAT-luciferase reporter gene activity in the absence of TRPC6. In line with these findings inhibition of TRPC3 using the specific inhibitor Pyr3 significantly reduced intracellular calcium concentrations and normalized osteoclastic differentiation and resorptive activity of TRPC6-deficient cells. Interestingly, an up-regulation of TRPC3 could be detected in a cohort of patients with low bone mineral density by comparing micro array data sets of circulating human osteoclast precursor cells to those from patients with high bone mineral density, suggesting a noticeable contribution of TRP calcium channels on bone quality. These observations demonstrate a novel regulatory function of TRPC channels in the process of osteoclastic differentiation and bone loss

1,25-Dihydroxyvitamin D3 Treatment Delays Cellular Aging in Human Mesenchymal Stem Cells while Maintaining Their Multipotent Capacity

<div><p>1,25-dihydroxyvitamin D3 (1,25D3) was reported to induce premature organismal aging in <em>fibroblast growth factor-23</em> (<em>Fgf23</em>) and <em>klotho</em> deficient mice, which is of main interest as 1,25D3 supplementation of its precursor cholecalciferol is used in basic osteoporosis treatment. We wanted to know if 1,25D3 is able to modulate aging processes on a cellular level in human mesenchymal stem cells (hMSC). Effects of 100 nM 1,25D3 on hMSC were analyzed by cell proliferation and apoptosis assay, β-galactosidase staining, VDR and surface marker immunocytochemistry, RT-PCR of 1,25D3-responsive, quiescence- and replicative senescence-associated genes. 1,25D3 treatment significantly inhibited hMSC proliferation and apoptosis after 72 h and delayed the development of replicative senescence in long-term cultures according to β-galactosidase staining and <em>P16</em> expression. Cell morphology changed from a fibroblast like appearance to broad and rounded shapes. Long term treatment did not induce lineage commitment in terms of osteogenic pathways but maintained their clonogenic capacity, their surface marker characteristics (expression of CD73, CD90, CD105) and their multipotency to develop towards the chondrogenic, adipogenic and osteogenic pathways. In conclusion, 1,25D3 delays replicative senescence in primary hMSC while the pro-aging effects seen in mouse models might mainly be due to elevated systemic phosphate levels, which propagate organismal aging.</p> </div

Effects of 1,25D3 treatment on quiescence- and senescence-associated genes.

<p><b>A</b> Expression of quiescence markers and densitometric quantification of semiquantitative RT-PCR results of hMSC (P3) treated with or without 1,25D3. Cells from five different donors were used. For each donor the Fold Change was calculated by comparing the expression of 1,25D3 treated cells with control cells. The results are shown as mean+SEM. The gene expression levels of <i>FOXO1a</i>, <i>FOXO3a</i>, <i>FOXO4</i>, <i>NANOG</i>, <i>TxNIP</i>, <i>TP53</i> were induced by 1,25D3 treatment over 3 passages. (FOXO1a: forkhead box 1a, FOXO3a: forkhead box 3a, FOXO4: forkhead box 4, NANOG: Nanog homeobox, TxNIP: thioredoxin interacting protein, TP53: tumor protein p53). <b>B</b> Expression of senescence markers and densitometric quantification of semiquantitative RT-PCR results of hMSC treated with or without 1,25D3 over at least four passages. 1,25D3 treatment reduced <i>P16</i> expression (p<0.05) and induced <i>P15</i> expression in cells at P4 compared to control cells. <i>P27</i> and <i>P21</i> gene expression in P4 showed no (<i>P27</i>) or only very weak (<i>P21)</i> induction by 1,25D3 treatment. Densitometric quantification of the semi-quantitative PCR results revealed downregulation of <i>PSG1</i> and almost no change in the expression of <i>PSG5</i> at P4 in 1,25D3 stimulated hMSC. The Relative Fold change represents the factor of different gene expression levels in 1,25D3 treated hMSC versus control cells. Expression profile of senescence-associated genes was determined in P4. Cells from three different donors were used. For each donor the Fold Change was calculated by comparing the expression of 1,25D3 treated cells with control cells. The results are shown as mean+SEM. (*, p<0.05, student's t-test).</p

Human Platelet Lysate versus Fetal Calf Serum: These Supplements Do Not Select for Different Mesenchymal Stromal Cells

International audienceCulture medium of mesenchymal stromal cells (MSCs) is usually supplemented with either human platelet lysate (HPL) or fetal calf serum (FCS). Many studies have demonstrated that proliferation and cellular morphology are affected by these supplements – it is therefore important to determine if they favor outgrowth of different subpopulations and thereby impact on the heterogeneous composition of MSCs. We have isolated and expanded human bone marrow-derived MSCs in parallel with HPL or FCS and demonstrated that HPL significantly increases proliferation and leads to dramatic differences in cellular morphology. Remarkably, global DNA-methylation profiles did not reveal any significant differences. Even at the transcriptomic level, there were only moderate changes in pairwise comparison. Furthermore, the effects on proliferation, cytoskeletal organization, and focal adhesions were reversible by interchanging to opposite culture conditions. These results indicate that cultivation of MSCs with HPL or FCS has no systematic bias for specific cell types

Trilineage differentiation capacity of 1,25D3 pretreated hMSC.

<p><b>A</b> Chondrogenic, adipogenic and osteogenic differentiation of hMSC after 1,25D3 pretreatment over three passages. Alcian Blue staining of chondrogenic differentiated hMSC (upper panel), Oil Red O staining for intracellular lipid vesicles during adipogenic differentiation (middle panel) and Alizarin Red S staining of mineralized extracellular matrix after osteogenic differentiation (lower panel) are shown (n.d. = not differentiated; C = differentiated control; +1,25D3 = differentiated, cells pretreated for three passages with 1,25D3). The figure is representative for three independent experiments. The bar represents 100 µm and 10 µm, respectively as indicated. <b>B</b> Quantification of chondrogenic (black bar), adipogenic (dark gray bar) and osteogenic differentiation (light gray bar) after three passages 1,25D3 pretreatment in comparison to untreated cells. For each differentiation lineage the Fold Change was calculated by comparing the induction of differentiation of 1,25D3 pretreated cells with control cells. The results are shown as means of three independent experiments+SEM using three different preparations of hMSC. Each time 5 to 9 pictures of each differentiation experiment were analyzed using the AutMess tool of the software of AxioVision Rel. 4.6.</p