FGF23 directly inhibits osteoprogenitor differentiation in Dmp1-knockout mice.

Fibroblast growth factor 23 (FGF23) is a phosphate-regulating (Pi-regulating) hormone produced by bone. Hereditary hypophosphatemic disorders are associated with FGF23 excess, impaired skeletal growth, and osteomalacia. Blocking FGF23 became an effective therapeutic strategy in X-linked hypophosphatemia, but testing remains limited in autosomal recessive hypophosphatemic rickets (ARHR). This study investigates the effects of Pi repletion and bone-specific deletion of Fgf23 on bone and mineral metabolism in the dentin matrix protein 1-knockout (Dmp1KO) mouse model of ARHR. At 12 weeks, Dmp1KO mice showed increased serum FGF23 and parathyroid hormone levels, hypophosphatemia, impaired growth, rickets, and osteomalacia. Six weeks of dietary Pi supplementation exacerbated FGF23 production, hyperparathyroidism, renal Pi excretion, and osteomalacia. In contrast, osteocyte-specific deletion of Fgf23 resulted in a partial correction of FGF23 excess, which was sufficient to fully restore serum Pi levels but only partially corrected the bone phenotype. In vitro, we show that FGF23 directly impaired osteoprogenitors' differentiation and that DMP1 deficiency contributed to impaired mineralization independent of FGF23 or Pi levels. In conclusion, FGF23-induced hypophosphatemia is only partially responsible for the bone defects observed in Dmp1KO mice. Our data suggest that combined DMP1 repletion and FGF23 blockade could effectively correct ARHR-associated mineral and bone disorders.

Bone-derived C-terminal FGF23 cleaved peptides increase iron availability in acute inflammation.

Inflammation leads to functional iron deficiency by increasing the expression of the hepatic iron regulatory peptide hepcidin. Inflammation also stimulates fibroblast growth factor 23 (FGF23) production by increasing both Fgf23 transcription and FGF23 cleavage, which paradoxically leads to excess in C-terminal FGF23 peptides (Cter-FGF23), rather than intact FGF23 (iFGF23) hormone. We determined that the major source of Cter-FGF23 is osteocytes and investigated whether Cter-FGF23 peptides play a direct role in the regulation of hepcidin and iron metabolism in response to acute inflammation. Mice harboring an osteocyte-specific deletion of Fgf23 showed a ∼90% reduction in Cter-FGF23 levels during acute inflammation. Reduction in Cter-FGF23 led to a further decrease in circulating iron in inflamed mice owing to excessive hepcidin production. We observed similar results in mice showing impaired FGF23 cleavage owing to osteocyte-specific deletion of Furin. We next showed that Cter-FGF23 peptides bind members of the bone morphogenetic protein (BMP) family, BMP2 and BMP9, which are established inducers of hepcidin. Coadministration of Cter-FGF23 and BMP2 or BMP9 prevented the increase in Hamp messenger RNA and circulating hepcidin levels induced by BMP2/9, resulting in normal serum iron levels. Finally, injection of Cter-FGF23 in inflamed Fgf23KO mice and genetic overexpression of Cter-Fgf23 in wild type mice also resulted in lower hepcidin and higher circulating iron levels. In conclusion, during inflammation, bone is the major source of Cter-FGF23 secretion, and independently of iFGF23, Cter-FGF23 reduces BMP-induced hepcidin secretion in the liver.

ZEB1 promotes pathogenic Th1 and Th17 cell differentiation in multiple sclerosis.

Inappropriate CD4+ T helper (Th) differentiation can compromise host immunity or promote autoimmune disease. To identify disease-relevant regulators of T cell fate, we examined mutations that modify risk for multiple sclerosis (MS), a canonical organ-specific autoimmune disease. This analysis identified a role for Zinc finger E-box-binding homeobox (ZEB1). Deletion of ZEB1 protects against experimental autoimmune encephalitis (EAE), a mouse model of multiple sclerosis (MS). Mechanistically, ZEB1 in CD4+ T cells is required for pathogenic Th1 and Th17 differentiation. Genomic analyses of paired human and mouse expression data elucidated an unexpected role for ZEB1 in JAK-STAT signaling. ZEB1 inhibits miR-101-3p that represses JAK2 expression, STAT3/STAT4 phosphorylation, and subsequent expression of interleukin-17 (IL-17) and interferon gamma (IFN-γ). Underscoring its clinical relevance, ZEB1 and JAK2 downregulation decreases pathogenic cytokines expression in T cells from MS patients. Moreover, a Food and Drug Administration (FDA)-approved JAK2 inhibitor is effective in EAE. Collectively, these findings identify a conserved, potentially targetable mechanism regulating disease-relevant inflammation.

Unraveling the intracellular efficacy of dextran-histidine polycation as an efficient nonviral gene delivery system.

In this study, we attempted to elucidate the capability of a natural polymer dextran, by modification with histidine, to be an efficient, safe and promising nucleic acid delivery system in gene therapy. Physicochemical characterizations were performed to get an insight into the derivative. The efficiency of the derivative as a gene delivery vehicle was also studied in depth using fluorescence microscopy. Extensive efforts were made to have a better understanding of the cellular dynamics involved. The derivative proved itself to be 6.7-fold more excelling than PEI in its transfecting capability. Mechanisms underlying cellular internalization, vector unpacking, intranuclear localization and transgene expression were also investigated. The possibility of recruiting intracellular histone to promote the entry of the gene into the nucleus seemed promising. Our findings also explored the links that mediate the correlation between the uptake of the derivative and various endocytic pathways. The results thus obtained reflect the success of the entire journey of the synthesized delivery vehicle.

Dextran-protamine polycation: an efficient nonviral and haemocompatible gene delivery system.

Despite the remarkable progress in the field of gene therapy with viral vectors, nonviral vectors have attracted great interests due to their unique properties. Imparting desired characteristics to nonviral gene delivery systems requires the development of cationic polymers. The purpose of this work was to design a cationic derivative (Dex-P) of dextran using protamine in order to assert target specific cellular binding. Our objective was to elucidate the potential use of Dex-P as a haemocompatible, nontoxic and efficient nonviral candidate for gene therapy. Nanoplexes were prepared with calf thymus DNA and Dex-P. Derivatization was confirmed by FTIR, gel permeation chromatography and TNBS assay. Dynamic light scattering and TEM studies determined the size and morphology of the nanoplex. The buffering behaviour was assessed by acid base titration. Complexation stability was evaluated using agarose gel electrophoresis and EtBr displacement assay. The protection of ctDNA from nuclear digestion and the effect of plasma components towards stability of the nanoplexes were also analyzed. Various haemocompatible studies were performed to check haemolysis, aggregation, clotting time, and complement activation. Transfection and cytotoxicity experiments were performed in vitro. The nanosize, spherical shape and stability of nanoplexes were affirmed. Various experiments conducted confirmed Dex-P to be nontoxic and haemocompatible. Transfection experiments revealed the capability of Dex-P to facilitate high gene expression and cellular uptake in HepG2 cells. With the improved physicochemical, biological and transfection properties, Dex-P seems to be a promising gene delivery system.

Dextran-glycidyltrimethylammonium chloride conjugate/DNA nanoplex: A potential non-viral and haemocompatible gene delivery system.

Non-viral gene carriers have attracted great interests for their unique properties. Cationic polymers have been in focus nowadays. Dextran is one of the most widely studied polymer in terms of gene therapy and in vivo disposition. But its applications are limited by its own drawbacks. To overcome the drawback, we have modified dextran using glycidyltrimethylammonium chloride (GTAC) bearing cationic groups. Nanoplexes were prepared using the derivative and calf thymus DNA (ctDNA) by reducing the surface charge and size of ctDNA. Complexation and stability of the nanoplex was proved using agarose gel electrophoresis and by Ethidium bromide (EtBr) displacement assay. Acid base titration studies were done to determine its buffering capacity. Derivatization was confirmed using NMR. Protection of ctDNA from nuclease digestion was evaluated. Stability of the nanoplex towards plasma components was analyzed. Its interactions with blood components were tested by haemolysis and aggregation studies. In vitro cytotoxicity studies have been done to investigate the effect of nanoplex on HepG2 cells by MTT assay. This derivative has been proved to be feasible in transfection. The above investigations prove the capability of dextran modified with GTAC as a promising non-viral and haemocompatible gene delivery agent.