Newly identified roles for PIEZO1 mechanosensor in controlling normal megakaryocyte development and in primary myelofibrosis.

Mechanisms through which mature megakaryocytes (Mks) and their progenitors sense the bone marrow extracellular matrix to promote lineage differentiation in health and disease are still partially understood. We found PIEZO1, a mechanosensitive cation channel, to be expressed in mouse and human Mks. Human mutations in PIEZO1 have been described to be associated with blood cell disorders. Yet, a role for PIEZO1 in megakaryopoiesis and proplatelet formation has never been investigated. Here, we show that activation of PIEZO1 increases the number of immature Mks in mice, while the number of mature Mks and Mk ploidy level are reduced. Piezo1/2 knockout mice show an increase in Mk size and platelet count, both at basal state and upon marrow regeneration. Similarly, in human samples, PIEZO1 is expressed during megakaryopoiesis. Its activation reduces Mk size, ploidy, maturation, and proplatelet extension. Resulting effects of PIEZO1 activation on Mks resemble the profile in Primary Myelofibrosis (PMF). Intriguingly, Mks derived from Jak2V617F PMF mice show significantly elevated PIEZO1 expression, compared to wild-type controls. Accordingly, Mks isolated from bone marrow aspirates of JAK2V617F PMF patients show increased PIEZO1 expression compared to Essential Thrombocythemia. Most importantly, PIEZO1 expression in bone marrow Mks is inversely correlated with patient platelet count. The ploidy, maturation, and proplatelet formation of Mks from JAK2V617F PMF patients are rescued upon PIEZO1 inhibition. Together, our data suggest that PIEZO1 places a brake on Mk maturation and platelet formation in physiology, and its upregulation in PMF Mks might contribute to aggravating some hallmarks of the disease.

Novel ANO5 mutation c.1067G>T (p.C356F) identified by whole genome sequencing in a big family with atypical gnathodiaphyseal dysplasia.

BACKGROUND: Gnathodiaphyseal dysplasia (GDD) is a rare skeletal disorder that has not been well studied. METHODS: Sanger sequencing, whole-genome sequencing (WGS), and bioinformatics and structural modeling analyses were performed. RESULTS: A family with patients with fibro-osseous lesions of the jawbones were initially diagnosed with cherubism. Sequencing of SH3BP2, which is the causal gene of cherubism, revealed no pathogenic mutation. Through WGS, we identified a novel mutation c.1067G>T (p.C356F) in ANO5, and bioinformatics analyses and structural modeling showed that the mutation was deleterious. Because ANO5 is the gene responsible for GDD, we reappraised the clinical data of the patients, and the diagnosis was corrected to atypical GDD. A review of the literature showed that 67% of GDD cases confirmed by molecular testing were initially misdiagnosed. CONCLUSIONS: The novel mutation c.1067G>T (p.C356F) in ANO5 is responsible for the atypical GDD observed in our patients. GDD should be included in the differential diagnosis for patients with fibro-osseous lesions.

Immunization with cell-free-generated vaccine protects from Porphyromonas gingivalis-induced alveolar bone loss.

INTRODUCTION: Periodontal diseases (PD) are complex oral inflammatory diseases initiated by keystone bacteria such as Porphyromonas gingivalis. A vaccine for PD is desirable as clinical treatment involves protracted maintenance strategies aimed to retain dentition. Although prior immunization approaches targeting P. gingivalis have reported variable success in limiting facets of disease such as oral bone loss, it remains that a vaccine for this disease may be attainable. AIM: To investigate cell-free protein synthesis (CFPS) as a platform to produce vaccinable targets suitable for efficacy testing in a P. gingivalis-induced murine oral bone loss model. MATERIALS AND METHODS: Recombinantly generated P. gingivalis minor fimbriae protein (Mfa1), RgpA gingipain hemagglutinin domain 1 (HA1), and RgpA gingipain hemagglutinin domain 2 (HA2) were combined in equivalent doses in adjuvants and injected intramuscularly to immunize mice. Serum levels of protein-specific antibody were measured by ELISA, and oral bone levels were defined by morphometrics. RESULTS: Recombinantly generated P. gingivalis proteins possessed high fidelity to predicted size and elicited protein-specific IgG following immunization. Importantly, immunization with the vaccine cocktail protected from P. gingivalis elicited oral bone loss. CONCLUSION: These data verify the utility of the CFPS technology to synthesize proteins that have the capacity to serve as novel vaccines.

Inflammatory response to Porphyromonas gingivalis partially requires interferon regulatory factor (IRF) 3.

Innate immune activation with expression of pro-inflammatory molecules such as TNF-α is a hallmark of the chronic inflammation associated with periodontal disease (PD). Porphyromonas gingivalis, a bacterium associated with PD, engages TLRs and activates MyD88-dependent and TIR-domain-containing adapter-inducing IFN-ß (TRIF)-dependent signaling pathways. IFN regulatory factor (IRF) 3 is activated in a TRIF-dependent manner and participates in production of cytokines such as TNF-α; however, little is known regarding IRF3 and the host response to PD pathogens. We speculated that IRF3 participates in the host inflammatory response to P. gingivalis. Our results show that bone marrow macrophages (MØ) from WT mice respond to P. gingivalis with activation and nuclear translocation of IRF3. Compared with WT, MØ from IRF3(-/-), TRIF(-/-), and TLR4(-/-) mice responded with reduced levels of TNF-α on P. gingivalis challenge. In addition, full expression of IL-6 and RANTES by MØ to P. gingivalis was dependent on IRF3. Lastly, employing MØ from IRF3(-/-) and IRF7(-/-) mice we observed a significant role for IRF3 and a modest role for IRF7 in the P. gingivalis-elicited TNF-α response. These studies identify a role for IRF3 in the inflammatory response by MØ to the periodontal pathogen P. gingivalis.

Fatty acids are rapidly delivered to and extracted from membranes by methyl-beta-cyclodextrin.

We performed detailed biophysical studies of transfer of long-chain fatty acids (FAs) from methyl-beta-CD (MBCD) to model membranes (egg-PC vesicles) and cells and the extraction of FA from membranes by MBCD. We used i) fluorescein phosphatidylethanolamine to detect transfer of FA anions arriving in the outer membrane leaflet; ii) entrapped pH dyes to measure pH changes after FA diffusion (flip-flop) across the lipid bilayer; and iii) soluble fluorescent-labeled FA binding protein to measure the concentration of unbound FA in water. FA dissociated from MBCD, bound to the membrane, and underwent flip-flop within milliseconds. In the presence of vesicles, MBCD maintained the aqueous concentration of unbound FA at low levels comparable to those measured with albumin. In studies with cells, addition of oleic acid (OA) complexed with MBCD yielded rapid (seconds) dose-dependent OA transport into 3T3-L1 preadipocytes and HepG2 cells. MBCD extracted OA from cells and model membranes rapidly at concentrations exceeding those required for OA delivery but much lower than concentrations commonly used for extracting cholesterol. Compared with albumin, MBCD can transfer its entire FA load and is less likely to extract cell nutrients and to introduce impurities.

Fatty acid transport and metabolism in HepG2 cells.

The mechanism(s) of fatty acid uptake by liver cells is not fully understood. We applied new approaches to address long-standing controversies of fatty acid uptake and to distinguish diffusion and protein-based mechanisms. Using HepG2 cells containing an entrapped pH-sensing fluorescence dye, we showed that the addition of oleate (unbound or bound to cyclodextrin) to the external buffer caused a rapid (seconds) and dose-dependent decrease in intracellular pH (pH(in)), indicating diffusion of fatty acids across the plasma membrane. pH(in) returned to its initial value with a time course (in min) that paralleled the metabolism of radiolabeled oleate. Preincubation of cells with the inhibitors phloretin or triacsin C had no effect on the rapid pH(in) drop after the addition of oleate but greatly suppressed pH(in) recovery. Using radiolabeled oleate, we showed that its esterification was almost completely inhibited by phloretin or triacsin C, supporting the correlation between pH(in) recovery and metabolism. We then used a dual-fluorescence assay to study the interaction between HepG2 cells and cis-parinaric acid (PA), a naturally fluorescent but slowly metabolized fatty acid. The fluorescence of PA increased rapidly upon its addition to cells, indicating rapid binding to the plasma membrane; pH(in) decreased rapidly and simultaneously but did not recover within 5 min. Phloretin had no effect on the PA-mediated pH(in) drop or its slow recovery but decreased the absolute fluorescence of membrane-bound PA. Our results show that natural fatty acids rapidly bind to, and diffuse through, the plasma membrane without hindrance by metabolic inhibitors or by an inhibitor of putative membrane-bound fatty acid transporters.