A TNIP1-driven systemic autoimmune disorder with elevated IgG4.

Whole-exome sequencing of two unrelated kindreds with systemic autoimmune disease featuring antinuclear antibodies with IgG4 elevation uncovered an identical ultrarare heterozygous TNIP1Q333P variant segregating with disease. Mice with the orthologous Q346P variant developed antinuclear autoantibodies, salivary gland inflammation, elevated IgG2c, spontaneous germinal centers and expansion of age-associated B cells, plasma cells and follicular and extrafollicular helper T cells. B cell phenotypes were cell-autonomous and rescued by ablation of Toll-like receptor 7 (TLR7) or MyD88. The variant increased interferon-ß without altering nuclear factor kappa-light-chain-enhancer of activated B cells signaling, and impaired MyD88 and IRAK1 recruitment to autophagosomes. Additionally, the Q333P variant impaired TNIP1 localization to damaged mitochondria and mitophagosome formation. Damaged mitochondria were abundant in the salivary epithelial cells of Tnip1Q346P mice. These findings suggest that TNIP1-mediated autoimmunity may be a consequence of increased TLR7 signaling due to impaired recruitment of downstream signaling molecules and damaged mitochondria to autophagosomes and may thus respond to TLR7-targeted therapeutics.

Glutathione transferase Omega 1 confers protection against azoxymethane-induced colorectal tumour formation.

Inflammatory bowel disease (IBD) is characterized by multiple alterations in cytokine expression and is a risk factor for colon cancer. The Omega class glutathione transferase GSTO1-1 regulates the release of the pro-inflammatory cytokines interleukin 1ß (IL-1ß) and interleukin 18 (IL-18) by deglutathionylating NEK7 in the NLRP3 inflammasome. When treated with azoxymethane and dextran sodium sulphate (AOM/DSS) as a model of IBD, Gsto1-/- mice were highly sensitive to colitis and showed a significant increase in the size and number of colon tumours compared with wild-type (WT) mice. Gsto1-/- mice treated with AOM/DSS had significantly lower serum IL-1ß and IL-18 levels as well as significantly decreased interferon (IFN)-γ, decreased pSTAT1 and increased pSTAT3 levels in the distal colon compared with similarly treated WT mice. Histologically, AOM/DSS treated Gsto1-/- mice showed increased active chronic inflammation with macrophage infiltration, epithelial dysplasia and invasive adenocarcinoma compared with AOM/DSS treated WT mice. Thus, this study shows that GSTO1-1 regulates IL-1ß and IL-18 activation and protects against colorectal cancer formation in the AOM/DSS model of IBD. The data suggest that while GSTO1-1 is a new target for the regulation of the NLRP3 inflammasome-associated cytokines IL-1ß and IL-18 by small molecule inhibitors, there is a possibility that anti-inflammatory drugs targeting these cytokines may potentiate colon cancer in some situations.

Prophylactic Herpes Simplex Virus 2 (HSV-2) Vaccines Adjuvanted with Stable Emulsion and Toll-Like Receptor 9 Agonist Induce a Robust HSV-2-Specific Cell-Mediated Immune Response, Protect against Symptomatic Disease, and Reduce the Latent Viral Reservoir.

Several prophylactic vaccines targeting herpes simplex virus 2 (HSV-2) have failed in the clinic to demonstrate sustained depression of viral shedding or protection from recurrences. Although these vaccines have generated high titers of neutralizing antibodies (NAbs), their induction of robust CD8 T cells has largely been unreported, even though evidence for the importance of HSV-2 antigen-specific CD8 T cells is mounting in animal models and in translational studies involving subjects with active HSV-2-specific immune responses. We developed a subunit vaccine composed of the NAb targets gD and gB and the novel T cell antigen and tegument protein UL40, and we compared this vaccine to a whole-inactivated-virus vaccine (formaldehyde-inactivated HSV-2 [FI-HSV-2]). We evaluated different formulations in combination with several Th1-inducing Toll-like receptor (TLR) agonists in vivo In mice, the TLR9 agonist cytosine-phosphate-guanine (CpG) oligodeoxynucleotide formulated in a squalene-based oil-in-water emulsion promoted most robust, functional HSV-2 antigen-specific CD8 T cell responses and high titers of neutralizing antibodies, demonstrating its superiority to vaccines adjuvanted by monophosphoryl lipid A (MPL)-alum. We further established that FI-HSV-2 alone or in combination with adjuvants as well as adjuvanted subunit vaccines were successful in the induction of NAbs and T cell responses in guinea pigs. These immunological responses were coincident with a suppression of vaginal HSV-2 shedding, low lesion scores, and a reduction in latent HSV-2 DNA in dorsal root ganglia to undetectable levels. These data support the further preclinical and clinical development of prophylactic HSV-2 vaccines that contain appropriate antigen and adjuvant components responsible for programming elevated CD8 T cell responses.IMPORTANCE Millions of people worldwide are infected with herpes simplex virus 2 (HSV-2), and to date, an efficacious prophylactic vaccine has not met the rigors of clinical trials. Attempts to develop a vaccine have focused primarily on glycoproteins necessary for HSV-2 entry as target antigens and to which the dominant neutralizing antibody response is directed during natural infection. Individuals with asymptomatic infection have exhibited T cell responses against specific HSV-2 antigens not observed in symptomatic individuals. We describe for the first time the immunogenicity profile in animal models of UL40, a novel HSV-2 T cell antigen that has been correlated with asymptomatic HSV-2 disease. Additionally, vaccine candidates adjuvanted by a robust formulation of the CpG oligonucleotide delivered in emulsion were superior to unadjuvanted or MPL-alum-adjuvanted formulations at eliciting a robust cell-mediated immune response and blocking the establishment of a latent viral reservoir in the guinea pig challenge model of HSV-2 infection.

Dichloroacetate Prevents Cisplatin-Induced Nephrotoxicity without Compromising Cisplatin Anticancer Properties.

Cisplatin is an effective anticancer drug; however, cisplatin use often leads to nephrotoxicity, which limits its clinical effectiveness. In this study, we determined the effect of dichloroacetate, a novel anticancer agent, in a mouse model of cisplatin-induced AKI. Pretreatment with dichloroacetate significantly attenuated the cisplatin-induced increase in BUN and serum creatinine levels, renal tubular apoptosis, and oxidative stress. Additionally, pretreatment with dichloroacetate accelerated tubular regeneration after cisplatin-induced renal damage. Whole transcriptome sequencing revealed that dichloroacetate prevented mitochondrial dysfunction and preserved the energy-generating capacity of the kidneys by preventing the cisplatin-induced downregulation of fatty acid and glucose oxidation, and of genes involved in the Krebs cycle and oxidative phosphorylation. Notably, dichloroacetate did not interfere with the anticancer activity of cisplatin in vivo. These data provide strong evidence that dichloroacetate preserves renal function when used in conjunction with cisplatin.

The transcription factor neural retina leucine zipper (NRL) controls photoreceptor-specific expression of myocyte enhancer factor Mef2c from an alternative promoter.

Neural retina leucine zipper (NRL) is an essential transcription factor for cell fate specification and functional maintenance of rod photoreceptors in the mammalian retina. In the Nrl(-/-) mouse retina, photoreceptor precursors fail to produce rods and generate functional cone photoreceptors that predominantly express S-opsin. Previous global expression analysis using microarrays revealed dramatically reduced expression of myocyte enhancer factor Mef2c in the adult Nrl(-/-) retina. We undertook this study to examine the biological relevance of Mef2c expression in retinal rod photoreceptors. Bioinformatics analysis, rapid analysis of cDNA ends (5'-RACE), and reverse transcription coupled with qPCR using splice site-specific oligonucleotides suggested that Mef2c is expressed in the mature retina from an alternative promoter. Chromatin immunoprecipitation (ChIP) studies showed the association of active RNA polymerase II and acetylated histone H3 just upstream of Mef2c exon 4, providing additional evidence for the utilization of an alternative promoter in the retina. In concordance, we observed the binding of NRL to a putative NRL-response element (NRE) at this location by ChIP-seq and electrophoretic mobility shift assays. NRL also activated the Mef2c alternative promoter in vitro and in vivo. Notably, MEF2C could support Rhodopsin promoter activity in rod photoreceptors. We conclude that Mef2c expression from an alternative promoter in the retina is regulated by NRL. Our studies also implicate MEF2C as a transcriptional regulator of homeostasis in rod photoreceptor cells.

Primary cilium-dependent mechanosensing is mediated by adenylyl cyclase 6 and cyclic AMP in bone cells.

Primary cilia are chemosensing and mechanosensing organelles that regulate remarkably diverse processes in a variety of cells. We previously showed that primary cilia play a role in mediating mechanosensing in bone cells through an unknown mechanism that does not involve extracellular Ca(2+)-dependent intracellular Ca(2+) release, which has been implicated in all other cells that transduce mechanical signals via the cilium. Here, we identify a molecular mechanism linking primary cilia and bone cell mechanotransduction that involves adenylyl cyclase 6 (AC6) and cAMP. Intracellular cAMP was quantified in MLO-Y4 cells exposed to dynamic flow, and AC6 and primary cilia were inhibited using RNA interference. When exposed to flow, cells rapidly (<2 min) and transiently decreased cAMP production in a primary cilium-dependent manner. RT-PCR revealed differential expression of the membrane-bound isoforms of adenylyl cyclase, while immunostaining revealed one, AC6, preferentially localized to the cilium. Further studies showed that decreases in cAMP in response to flow were dependent on AC6 and Gd(3+)-sensitive channels but not intracellular Ca(2+) release and that this response mediated flow-induced COX-2 gene expression. The signaling events identified provide important details of a novel early mechanosensing mechanism in bone and advances our understanding of how signal transduction occurs at the primary cilium.

Temporal ChIP-on-Chip of RNA-Polymerase-II to detect novel gene activation events during photoreceptor maturation.

PURPOSE: During retinal development, post-mitotic neural progenitor cells must activate thousands of genes to complete synaptogenesis and terminal maturation. While many of these genes are known, others remain beyond the sensitivity of expression microarray analysis. Some of these elusive gene activation events can be detected by mapping changes in RNA polymerase-II (Pol-II) association around transcription start sites. METHODS: High-resolution (35 bp) chromatin immunoprecipitation (ChIP)-on-chip was used to map changes in Pol-II binding surrounding 26,000 gene transcription start sites during photoreceptor maturation of the mouse neural retina, comparing postnatal age 25 (P25) to P2. Coverage was 10-12 kb per transcription start site, including 2.5 kb downstream. Pol-II-active regions were mapped to the mouse genomic DNA sequence by using computational methods (Tiling Analysis Software-TAS program), and the ratio of maximum Pol-II binding (P25/P2) was calculated for each gene. A validation set of 36 genes (3%), representing a full range of Pol-II signal ratios (P25/P2), were examined with quantitative ChIP assays for transcriptionally active Pol-II. Gene expression assays were also performed for 19 genes of the validation set, again on independent samples. FLT-3 Interacting Zinc-finger-1 (FIZ1), a zinc-finger protein that associates with active promoter complexes of photoreceptor-specific genes, provided an additional ChIP marker to highlight genes activated in the mature neural retina. To demonstrate the use of ChIP-on-chip predictions to find novel gene activation events, four additional genes were selected for quantitative PCR analysis (qRT-PCR analysis); these four genes have human homologs located in unidentified retinal disease regions: Solute carrier family 25 member 33 (Slc25a33), Lysophosphatidylcholine acyltransferase 1 (Lpcat1), Coiled-coil domain-containing 126 (Ccdc126), and ADP-ribosylation factor-like 4D (Arl4d). RESULTS: ChIP-on-chip Pol-II peak signal ratios >1.8 predicted increased amounts of transcribing Pol-II and increased expression with an estimated 97% accuracy, based on analysis of the validation gene set. Using this threshold ratio, 1,101 genes were predicted to experience increased binding of Pol-II in their promoter regions during terminal maturation of the neural retina. Over 800 of these gene activations were additional to those previously reported by microarray analysis. Slc25a33, Lpcat1, Ccdc126, and Arl4d increased expression significantly (p<0.001) during photoreceptor maturation. Expression of all four genes was diminished in adult retinas lacking rod photoreceptors (Rd1 mice) compared to normal retinas (90% loss for Ccdc126 and Arl4d). For rhodopsin (Rho), a marker of photoreceptor maturation, two regions of maximum Pol-II signal corresponded to the upstream rhodopsin enhancer region and the rhodopsin proximal promoter region. CONCLUSIONS: High-resolution maps of Pol-II binding around transcription start sites were generated for the postnatal mouse retina; which can predict activation increases for a specific gene of interest. Novel gene activation predictions are enriched for biologic functions relevant to vision, neural function, and chromatin regulation. Use of the data set to detect novel activation increases was demonstrated by expression analysis for several genes that have human homologs located within unidentified retinal disease regions: Slc25a33, Lpcat1, Ccdc126, and Arl4d. Analysis of photoreceptor-deficient retinas indicated that all four genes are expressed in photoreceptors. Genome-wide maps of Pol-II binding were developed for visual access in the University of California, Santa Cruz (UCSC) Genome Browser and its eye-centric version EyeBrowse (National Eye Institute-NEI). Single promoter resolution of Pol-II distribution patterns suggest the Rho enhancer region and the Rho proximal promoter region become closely associated with the activated gene's promoter complex.

Beta1 integrins mediate mechanosensitive signaling pathways in osteocytes.

Integrins are cell-substrate adhesion proteins that initiate intracellular signaling and may serve as mechanosensors in bone. MLO-Y4 cells were stably transfected with a dominant negative form of the beta(1) integrin subunit (beta(1)DN) containing the transmembrane domain and cytoplasmic tail of beta(1) integrin. Cells expressing beta(1)DN had reduced vinculin localization to focal contacts but no change in intracellular actin organization. When exposed to oscillatory fluid flow, beta(1)DN cells exhibited a significant reduction in the upregulation of cyclooxygenase-2 gene expression and prostaglandin E(2) release. Similarly, the ratio of receptor activator of NF-kappaB ligand mRNA to osteoprotegerin mRNA decreased significantly after exposure to fluid flow in control cells but not in beta(1)DN cells. Interfering with integrin signaling did not affect mechanically induced intracellular calcium mobilization. These data suggest that integrins may initiate the cellular response of osteocytes to dynamic fluid flow and may serve as mechanosensitive molecules in bone.

Development of Benzenesulfonamide Derivatives as Potent Glutathione Transferase Omega-1 Inhibitors.

Glutathione transferase omega-1 (GSTO1-1) is an enzyme whose function supports the activation of interleukin (IL)-1ß and IL-18 that are implicated in a variety of inflammatory disease states for which small-molecule inhibitors are sought. The potent reactivity of the active-site cysteine has resulted in reported inhibitors that act by covalent labeling. In this study, structure-activity relationship (SAR) elaboration of the reported GSTO1-1 inhibitor C1-27 was undertaken. Compounds were evaluated for inhibitory activity toward purified recombinant GSTO1-1 and for indicators of target engagement in cell-based assays. As covalent inhibitors, the kinact/KI values of selected compounds were determined, as well as in vivo pharmacokinetics analysis. Cocrystal structures of key novel compounds in complex with GSTO1-1 were also solved. This study represents the first application of a biochemical assay for GSTO1-1 to determine kinact/KI values for tested inhibitors and the most extensive set of cell-based data for a GSTO1-1 inhibitor SAR series reported to date. Our research culminated in the discovery of 25, which we propose as the preferred biochemical tool to interrogate cellular responses to GSTO1-1 inhibition.

Glutathione Transferase Omega-1 Regulates NLRP3 Inflammasome Activation through NEK7 Deglutathionylation.

The NLRP3 inflammasome is a cytosolic complex sensing phagocytosed material and various damage-associated molecular patterns, triggering production of the pro-inflammatory cytokines interleukin-1 beta (IL)-1ß and IL-18 and promoting pyroptosis. Here, we characterize glutathione transferase omega 1-1 (GSTO1-1), a constitutive deglutathionylating enzyme, as a regulator of the NLRP3 inflammasome. Using a small molecule inhibitor of GSTO1-1 termed C1-27, endogenous GSTO1-1 knockdown, and GSTO1-1-/- mice, we report that GSTO1-1 is involved in NLRP3 inflammasome activation. Mechanistically, GSTO1-1 deglutathionylates cysteine 253 in NIMA related kinase 7 (NEK7) to promote NLRP3 activation. We therefore identify GSTO1-1 as an NLRP3 inflammasome regulator, which has potential as a drug target to limit NLRP3-mediated inflammation.

Osteocytes as mechanosensors in the inhibition of bone resorption due to mechanical loading.

Bone has the ability to adjust its structure to meet its mechanical environment. The prevailing view of bone mechanobiology is that osteocytes are responsible for detecting and responding to mechanical loading and initiating the bone adaptation process. However, how osteocytes signal effector cells and initiate bone turnover is not well understood. Recent in vitro studies have shown that osteocytes support osteoclast formation and activation when co-cultured with osteoclast precursors. In this study, we examined the osteocytes' role in the mechanical regulation of osteoclast formation and activation. We demonstrated here that (1) mechanical stimulation of MLO-Y4 osteocyte-like cells decreases their osteoclastogenic-support potential when co-cultured with RAW264.7 monocyte osteoclast precursors; (2) soluble factors released by these mechanically stimulated MLO-Y4 cells inhibit osteoclastogenesis induced by ST2 bone marrow stromal cells or MLO-Y4 cells; and (3) soluble RANKL and OPG were released by MLO-Y4 cells, and the expressions of both were found to be mechanically regulated. Our data suggest that mechanical loading decreases the osteocyte's potential to induce osteoclast formation by direct cell-cell contact. However, it is not clear that osteocytes in vivo are able to form contacts with osteoclast precursors. Our data also demonstrate that mechanically stimulated osteocytes release soluble factors that can inhibit osteoclastogenesis induced by other supporting cells including bone marrow stromal cells. In summary, we conclude that osteocytes may function as mechanotransducers by regulating local osteoclastogenesis via soluble signals.

Helicobacter pylori induces somatic mutations in TP53 via overexpression of CHAC1 in infected gastric epithelial cells.

Infection with Helicobacter pylori is known to decrease the level of glutathione in gastric epithelial cells and increase the production of reactive oxygen species (ROS), which can lead to DNA damage and the development of gastric cancer. Cation transport regulator 1 (CHAC1) has γ-glutamylcyclotransferase activity that degrades glutathione. We found that cagA-positive H. pylori infection triggered CHAC1 overexpression in human gastric epithelial (AGS) cells leading to glutathione degradation and the accumulation of ROS. Nucleotide alterations in the TP53 tumour suppressor gene were induced in AGS cells overexpressing CHAC1, whereas no mutations were detected in cells overexpressing a catalytically inactive mutant of CHAC1. A high frequency of TP53 mutations occurred in H. pylori-infected AGS cells, but this was prevented in cells transfected with CHAC1 siRNA. These findings indicate that H. pylori-mediated CHAC1 overexpression degrades intracellular glutathione, allowing the accumulation of ROS which subsequently causes mutations that could contribute to the development of gastric cancer.

GSTO1-1 plays a pro-inflammatory role in models of inflammation, colitis and obesity.

Glutathione transferase Omega 1 (GSTO1-1) is an atypical GST reported to play a pro-inflammatory role in response to LPS. Here we show that genetic knockout of Gsto1 alters the response of mice to three distinct inflammatory disease models. GSTO1-1 deficiency ameliorates the inflammatory response stimulated by LPS and attenuates the inflammatory impact of a high fat diet on glucose tolerance and insulin resistance. In contrast, GSTO1-1 deficient mice show a more severe inflammatory response and increased escape of bacteria from the colon into the lymphatic system in a dextran sodium sulfate mediated model of inflammatory bowel disease. These responses are similar to those of TLR4 and MyD88 deficient mice in these models and confirm that GSTO1-1 is critical for a TLR4-like pro-inflammatory response in vivo. In wild-type mice, we show that a small molecule inhibitor that covalently binds in the active site of GSTO1-1 can be used to ameliorate the inflammatory response to LPS. Our findings demonstrate the potential therapeutic utility of GSTO1-1 inhibitors in the modulation of inflammation and suggest their possible application in the treatment of a range of inflammatory conditions.

Circulating growth hormone binding protein levels and mononuclear cell growth hormone receptor expression in uremia.

BACKGROUND: Resistance to growth hormone (GH) in end-stage renal disease (ESRD) causes growth retardation and muscle wasting. In humans, circulating GH binding protein (GHBP), the extracellular domain of the GH receptor that is shed into the circulation and is believed to reflect tissue GH receptor levels, is reduced in uremia and suggests that cellular GH receptor levels are correspondingly reduced. If true, this could be a cause of GH resistance. We set out to establish whether serum GHBP levels reflect cellular GH receptor levels and whether changes in serum GHBP levels are related to nutritional or inflammatory status. METHODS: GH receptor protein expression in peripheral blood mononuclear cells (PBMC) from 21 ESRD and 14 normal subjects were analyzed by fluorochrome flow cytometry. RESULTS: The GH receptor density and percent total PBMCs expressing the GH receptor were similar in the 2 groups, and there was no difference in percent GH receptor positive T or B cells or monocytes. In contrast, serum GHBP levels were 80% lower in ESRD. GHBP levels did not correlate with serum albumin, body mass index, or muscle mass but seemed to be partly related to the log serum C-reactive protein levels. CONCLUSIONS: Serum GHBP levels are markedly reduced in ESRD; this seems to occur independent of nutritional status and may in part be caused by inflammation. Because GH receptor expression on PBMC of ESRD and control subjects was similar, our findings argue against a reduction in GH receptor as a cause of GH resistance and the use of serum GHBP levels as a reliable marker of specific tissue GH receptor levels.

Glutathione transferase M2 variants inhibit ryanodine receptor function in adult mouse cardiomyocytes.

Release of Ca(2+) from the sarcoplasmic reticulum (SR) through the cardiac ryanodine receptor (RyR2) is an essential step in cardiac excitation-contraction coupling. Excess Ca(2+) release due to overactive RyR2 can cause arrhythmia that can lead to cardiac arrest. Fragments derived from the carboxy-terminal domain of human glutathione transferase M2 (GSTM2C) specifically inhibit RyR2 activity. Our aim was to further improve this inhibition by mutagenesis and to assess the therapeutic potential of GSTM2C based peptides to treat Ca(2+) release-based arrhythmia. We generated several mutant variants of the C-terminal fragment GSTM2C H5-8 and from those mutant proteins we identified two (RM13 and SM2) that exhibited significantly greater inhibition of cardiac SR Ca(2+) release and single RyR2 channel activity. Flow cytometry analysis showed that these two mutant proteins as well as GSTM2C H5-8 are taken up by isolated adult mouse cardiomyocytes without the aid of any additional compounds, Ca(2+) imaging and isolated cell contraction measurements revealed that GSTM2C H5-8, SM2 and RM13 reduce the SR Ca(2+) release rate and the fractional shortening of adult mouse cardiomyocytes, while importantly increasing the rate of Ca(2+) removal from the sarcoplasm. These observations indicate that peptides derived from GSTM2C inhibit RyR2 at a cellular level and thus they may provide the basis for a novel therapeutic agent to treat arrhythmia and heart attack.

Dichloroacetic acid up-regulates hepatic glutathione synthesis via the induction of glutamate-cysteine ligase.

Dichloroacetic acid (DCA) has potential for use in cancer therapy and the treatment of metabolic acidosis. However, DCA can create a deficiency of glutathione transferase Zeta (GSTZ1-1). Gstz1 knockout mice have elevated oxidative stress and low glutathione levels that increases their sensitivity to acetaminophen toxicity. As it is highly likely that patients that are treated with DCA will develop drug induced GSTZ1-1 deficiency we considered they could be at risk of elevated toxicity if they are exposed to other drugs that cause oxidative stress or consume glutathione (GSH). To test this hypothesis we treated mice with DCA and acetaminophen (APAP). Surprisingly, the mice pre-treated with DCA suffered less APAP-mediated hepatotoxicity than untreated mice. This protection is most likely due to an increased capacity for the liver to synthesize GSH, since DCA increased the expression and activity of glutamate-cysteine ligase GCL, the rate-limiting enzyme of GSH synthesis. Other pathways for acetaminophen disposal were unchanged or diminished by DCA. Pre-treatment with DCA may be of use in other settings where the maintenance of protective levels of GSH are required. However, DCA may lower the efficacy of drugs that rely on oxidative stress and the depletion of GSH to enhance their cytotoxicity or of drugs that are detoxified by GSH conjugation. Consequently, as the use of DCA in the clinic is likely to increase, it will be critical to evaluate the interactions of DCA with other drugs to ensure the combinations retain their efficacy and do not cause enhanced toxicity.

The Role of Primary Cilia in Mesenchymal Stem Cell Differentiation: A Pivotal Switch in Guiding Lineage Commitment.

Primary cilia are sensory organelles that have been shown to play a critical role in lineage commitment. It was our hypothesis that the primary cilium is necessary for chemically induced differentiation of human mesenchymal stem cells (MSC). To investigate this, polaris siRNA was used to inhibit the primary cilia and the mRNA levels of transcription factors Runx2, PPARgamma were measured by RT PCR as markers of osteogenic, adipogenic and chondrogenic differentiation, respectively. MSCs with inhibited primary cilia had significantly decreased basal mRNA expression levels of all three lineages specific transcription factors indicating that primary cilia are critical in multiple differentiation pathways. Furthermore, to determine if primary cilia play a role in the differentiation potential of MSCs, progenitor cells transfected with either scrambled or polaris siRNA were cultured in osteo-inductive, chondro-inductive, or adipo-inductive media and lineage commitment was ascertained. Interestingly, within 24 h of culture, cells transfected with polaris siRNA in both osteogenic and adipogenic media lost adhesion and released from the slides; however MSCs in chondrogenic media as well as cells transfected with scrambled siRNA did not. These results suggest that the primary cilium is necessary for the normal progression of chemically induced osteogenic and adipogenic differentiation. As a control, the experiment was repeated with NIH3T3 fibroblasts and none of the effects of inhibited primary cilia were observed indicating that the loss of adhesion may be specific to MSCs. Furthermore after biochemically inducing the cells to differentiate, polaris knockdown resulted in abrogation of both Runx2 and PPARgamma mRNA while SOX9 mRNA expression was significantly lower. These results suggest that primary cilia play an essential role not only in the initiation of both osteogenic and adipogenic differentiation, but also in maintaining the phenotype of differentiated cells. Interestingly, chondrogenic differentiation appeared less dependent on a functional primary cilium.

The epigenetic mechanism of mechanically induced osteogenic differentiation.

Epigenetic regulation of gene expression occurs due to alterations in chromatin proteins that do not change DNA sequence, but alter the chromatin architecture and the accessibility of genes, resulting in changes to gene expression that are preserved during cell division. Through this process genes are switched on or off in a more durable fashion than other transient mechanisms of gene regulation, such as transcription factors. Thus, epigenetics is central to cellular differentiation and stem cell linage commitment. One such mechanism is DNA methylation, which is associated with gene silencing and is involved in a cell's progression towards a specific fate. Mechanical signals are a crucial regulator of stem cell behavior and important in tissue differentiation; however, there has been no demonstration of a mechanism whereby mechanics can affect gene regulation at the epigenetic level. In this study, we identified candidate DNA methylation sites in the promoter regions of three osteogenic genes from bone marrow derived mesenchymal stem cells (MSCs). We demonstrate that mechanical stimulation alters their epigenetic state by reducing DNA methylation and show an associated increase in expression. We contrast these results with biochemically induced differentiation and distinguish expression changes associated with durable epigenetic regulation from those likely to be due to transient changes in regulation. This is an important advance in stem cell mechanobiology as it is the first demonstration of a mechanism by which the mechanical micro-environment is able to induce epigenetic changes that control osteogenic cell fate, and that can be passed to daughter cells. This is a first step to understanding that will be vital to successful bone tissue engineering and regenerative medicine, where continued expression of a desired long-term phenotype is crucial.

Non-canonical Wnt signaling and N-cadherin related beta-catenin signaling play a role in mechanically induced osteogenic cell fate.

BACKGROUND: Understanding how the mechanical microenvironment influences cell fate, and more importantly, by what molecular mechanisms, will enhance not only the knowledge of mesenchymal stem cell biology but also the field of regenerative medicine. Mechanical stimuli, specifically loading induced oscillatory fluid flow, plays a vital role in promoting healthy bone development, homeostasis and morphology. Recent studies suggest that such loading induced fluid flow has the potential to regulate osteogenic differentiation via the upregulation of multiple osteogenic genes; however, the molecular mechanisms involved in the transduction of a physical signal into altered cell fate have yet to be determined. METHODS AND PRINCIPAL FINDINGS: Using immuno-staining, western blot analysis and luciferase assays, we demonstrate the oscillatory fluid flow regulates beta-catenin nuclear translocation and gene transcription. Additionally, real time RT-PCR analysis suggests that flow induces Wnt5a and Ror2 upregulation, both of which are essential for activating the small GTPase, RhoA, upon flow exposure. Furthermore, although beta-catenin phosphorylation is not altered by flow, its association with N-cadherin is, indicating that flow-induced beta-catenin signaling is initiated by adherens junction signaling. CONCLUSION: We propose that the mechanical microenvironment of bone has the potential to regulate osteogenic differentiation by initiating multiple key molecular pathways that are essential for such lineage commitment. Specifically, non-canonical Wnt5a signaling involving Ror2 and RhoA as well as N-cadherin mediated beta-catenin signaling are necessary for mechanically induced osteogenic differentiation.

Mechanically induced osteogenic differentiation--the role of RhoA, ROCKII and cytoskeletal dynamics.

Many biochemical factors regulating progenitor cell differentiation have been examined in detail; however, the role of the local mechanical environment on stem cell fate has only recently been investigated. In this study, we examined whether oscillatory fluid flow, an exogenous mechanical signal within bone, regulates osteogenic, adipogenic or chondrogenic differentiation of C3H10T1/2 murine mesenchymal stem cells by measuring Runx2, PPARgamma and SOX9 gene expression, respectively. Furthermore, we hypothesized that the small GTPase RhoA and isometric tension within the actin cytoskeleton are essential in flow-induced differentiation. We found that oscillatory fluid flow induces the upregulation of Runx2, Sox9 and PPARgamma, indicating that it has the potential to regulate transcription factors involved in multiple unique lineage pathways. Furthermore, we demonstrate that the small GTPase RhoA and its effector protein ROCKII regulate fluid-flow-induced osteogenic differentiation. Additionally, activated RhoA and fluid flow have an additive effect on Runx2 expression. Finally, we show RhoA activation and actin tension are negative regulators of both adipogenic and chondrogenic differentiation. However, an intact, dynamic actin cytoskeleton under tension is necessary for flow-induced gene expression.