Physiological tonicity improves human chondrogenic marker expression through nuclear factor of activated T-cells 5 in vitro.
"Chondrocytes experience a hypertonic environment compared with plasma (280 mOsm) due to the high fixed negative charge density of cartilage. Standard isolation of chondrocytes removes their hypertonic matrix, exposing them to nonphysiological conditions. During in vitro expansion, chondrocytes quickly lose their specialized phenotype. We aimed to elucidate the effects of tonicity during isolation and in vitro expansion on chondrocyte phenotype.
Human articular chondrocytes were isolated and subsequently expanded at control tonicity (280 mOsm) or at moderately elevated, physiological tonicity (380 mOsm). The effects of physiological tonicity on chondrocyte proliferation and chondrogenic marker expression were evaluated. The role of Tonicity-responsive Enhancer Binding Protein in response to physiological tonicity was investigated using nuclear factor of activated T-cells 5 (NFAT5) RNA interference.
Moderately elevated, physiological tonicity (380 mOsm) did not affect chondrocyte proliferation, while higher tonicities inhibited proliferation and diminished cell viability. Physiological tonicity improved expression of chondrogenic markers and NFAT5 and its target genes, while suppressing dedifferentiation marker collagen type I and improving type II/type I expression ratios >100-fold. Effects of physiological tonicity were similar in osteoarthritic and normal (nonosteoarthritic) chondrocytes, indicating a disease-independent mechanism. NFAT5 RNA interference abolished tonicity-mediated effects and revealed that NFAT5 positively regulates collagen type II expression, while suppressing type I."
So upregulating NFAT5 may be a way to enhance LSJL results.
"Following NFAT5 knockdown, the NFAT5 targets S100A4[upregulated by LSJL] and SLC6A12 were also no longer hypertonically inducible: S100A4 expression decreased twofold and SLC6A12 was virtually undetectable upon NFAT5 RNAi "<-Despite a regulation in NFAT5 LSJL upregulated S100A4.
"Hypertonicity induces cell shrinkage, which may activate Na+, K+, or 2Cl- co-transport, allowing cellular accumulation of NaCl and KCl."
"Rho-type small G proteins and p38 kinases might also act upstream of NFAT5 in chondrocytes."
"p38 MAPK plays important roles in chondrocytes and seems to be necessary for NFAT5 expression"
"the promoter regions of both collagen type II and AGC1 contain a plethora of potential other binding sites for transcriptional enhancers and suppressors, such as SOX5/6, Barx2[up in LSJL], β-catenin, c-Maf, PIAS, TRAP230, Bapx1, and C/EBP and NF-κB "
"high NaCl rapidly activates p38 MAPK, its action can be isoform specific and may exert opposing effects on NFAT5"
Elevated extracellular glucose and uncontrolled type 1 diabetes enhance NFAT5 signaling and disrupt the transverse tubular network in mouse skeletal muscle., states that increasing extracellular glucose can stimulate NFAT5.
Differential regulation of NFAT5 by NKCC2 isoforms in medullary thick ascending limb (mTAL) cells., states that high NaCl may also upregulate NFAT5. "a 2.5-fold increase in NFAT5 mRNA accumulation was observed after cells were exposed to 500 mosmol/kgH₂O for 4 h. "
Transcriptional responses to intermittent hypoxia.
"IH[intermittent hypoxia] activates a variety of transcription factors including the hypoxia-inducible factor-1 (HIF-1), c-fos (immediate early gene), nuclear factor of activated T-cells (NFAT), and nuclear factor kB (NF-kB). The signaling pathways associated with transcriptional activation associated with IH differ from continuous hypoxia (CH). Compared to same duration and intensity of CH, IH is more potent in activating HIF-1 and c-fos and also results in long-lasting accumulation of HIF-1alpha and c-fos mRNA, a phenomenon that was not seen with CH. IH-evoked transcriptional activation by HIF-1, c-fos[up in LSJL] as well as the resulting activator protein-1 (AP-1) requires reactive oxygen species (ROS)-mediated signaling and involves complex feed forward interactions between HIF-1 and ROS. Chronic IH-evoked cardio-respiratory responses are absent in Hif-1alpha+/- mice, and hypertension elicited by chronic IH is absent in mice lacking [NFATC3]."
"Transcriptional activators that are affected by continuous hypoxia (CH) include: hypoxia inducible factors (HIF-1 and HIF-2); nuclear factor kappa B (NF-kB), cyclic AMP response element binding protein (CREB), activating protein-1 (AP-1), p53, early growth response-1 (Egr-1), nuclear factor for interleukin 6 (NF-IL6)"
"Although MAPKs (ERK-1 &2; Jun Kinase) are activated by IH, inhibitors of MAPKs and PI-3 kinase were ineffective in blocking IH-elicited activation of HIF-1 mediated transcriptional activation"
"chronic IH increases ROS in several tissues"
"ET-1[upregulated by LSJL as Edn1] is a potent activator of NFAT "
NFATC3 elevated mice blood pressure.
"Nuclear factor of activated T-cells (NFAT) and calcineurin are essential regulators of immune cell and mesenchymal cell differentiation. Elevated intracellular calcium induces chondrogenesis through a calcineurin/NFAT signaling axis that activates bone morphogenetic protein (BMP) expression. The calcium ionophore, ionomycin, induced chondrogenesis through activation of calcineurin. The calcineurin substrate, NFAT4, also induced chondrogenesis and chondrocyte gene expression. Significantly, the BMP antagonist, noggin, or dominant negative BMP receptors blocked the effects of elevated intracellular calcium on chondrogenesis. calcineurin/NFAT4 activates BMP expression. Consistent with this, BMP2 gene expression was increased by ionomycin and suppressed by the calcineurin inhibitor, cyclosporine A. Activated NFAT4[also known as NFATC3 which is downregulated by LSJL] induced BMP2 gene expression."
"Ionomycin produced a 2.5-fold increase in the number of Alcian blue staining nodules of cartilage. "
"Significantly, cyclosporine A (CsA) inhibited cartilage development both in the presence and absence of ionomycin "
"activated NFAT2 can induce cartilage gene expression"
"calcium-dependent signals induce expression of FGF18. The induction of FGF18 expression required the calcium-dependent phosphatase, calcineurin. The activated forms of calcineurin or the calcineurin-dependent transcription factor, NFAT4 (nuclear factor of activated T-cell 4), induced FGF18 expression. FGF18 or a constitutive active FGF receptor suppressed noggin gene induction and thereby increased chondrocyte gene expression and chondrogenesis by facilitating bone morphogenetic protein-dependent signals."
"FGF18 expression is induced up to 12-fold by the calcium ionophore, ionomycin "
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