Loss of Folliculin Disrupts Hematopoietic Stem Cell Quiescence and Homeostasis Resulting in Bone Marrow Failure.

Folliculin (FLCN) is an autosomal dominant tumor suppressor gene that modulates diverse signaling pathways required for growth, proliferation, metabolism, survival, motility, and adhesion. FLCN is an essential protein required for murine embryonic development, embryonic stem cell (ESC) commitment, and Drosophila germline stem cell maintenance, suggesting that Flcn may be required for adult stem cell homeostasis. Conditional inactivation of Flcn in adult hematopoietic stem/progenitor cells (HSPCs) drives hematopoietic stem cells (HSC) into proliferative exhaustion resulting in the rapid depletion of HSPC, loss of all hematopoietic cell lineages, acute bone marrow (BM) failure, and mortality after 40 days. HSC that lack Flcn fail to reconstitute the hematopoietic compartment in recipient mice, demonstrating a cell-autonomous requirement for Flcn in HSC maintenance. BM cells showed increased phosphorylation of Akt and mTorc1, and extramedullary hematopoiesis was significantly reduced by treating mice with rapamycin in vivo, suggesting that the mTorc1 pathway was activated by loss of Flcn expression in hematopoietic cells in vivo. Tfe3 was activated and preferentially localized to the nucleus of Flcn knockout (KO) HSPCs. Tfe3 overexpression in HSPCs impaired long-term hematopoietic reconstitution in vivo, recapitulating the Flcn KO phenotype, and supporting the notion that abnormal activation of Tfe3 contributes to the Flcn KO phenotype. Flcn KO mice develop an acute histiocytic hyperplasia in multiple organs, suggesting a novel function for Flcn in macrophage development. Thus, Flcn is intrinsically required to maintain adult HSC quiescence and homeostasis, and Flcn loss leads to BM failure and mortality in mice.

Functional analysis of Protection of Telomeres 1a (Pot1a) in regulation of hematopoietic stem cell aging.

Repeated cell divisions induce DNA damage accumulation, which impairs stem cell function during aging. However, the general molecular mechanisms by which this occurs remain unclear. Herein, we show that the expression of protection of telomeres 1a (Pot1a), a component of shelterin, is crucial for prevention of telomeric DNA damage response (DDR) and maintenance of hematopoietic stem cell (HSC) activity during aging. We observed that HSCs express high levels of Pot1a during development, and this expression declines with aging. Knockdown of Pot1a induced an age-related phenotype, characterized by increased telomeric DDR and reduced long-term reconstitution activity. In contrast, treatment with exogenous Pot1a protein prevented telomeric DDR, which decreased stem cell activity and partially rejuvenated HSC activity. These results highlight a general, reversible mechanism by which aging compromises mammalian stem cell activity, with widespread implications for regenerative medicine.

Prostaglandin E(2) regulates murine hematopoietic stem/progenitor cells directly via EP4 receptor and indirectly through mesenchymal progenitor cells.

Prostaglandin E(2) (PGE(2)) regulates hematopoietic stem/progenitor cell (HSPC) activity. However, the receptor(s) responsible for PGE(2) signaling remains unclear. Here, we identified EP4 as a receptor activated by PGE(2) to regulate HSPCs. Knockdown of Ep4 in HSPCs reduced long-term reconstitution capacity, whereas an EP4-selective agonist induced phosphorylation of GSK3ß and ß-catenin and enhanced long-term reconstitution capacity. Next, we analyzed the niche-mediated effect of PGE(2) in HSPC regulation. Bone marrow mesenchymal progenitor cells (MPCs) expressed EP receptors, and stimulation of MPCs with PGE(2) significantly increased their ability to support HSPC colony formation. Among the EP receptor agonists, only an EP4 agonist facilitated the formation of HSPC colonies after the coculture with MPCs. PGE(2) up-regulated the expression of cytokine-, cell adhesion-, extracellular matrix-, and protease-related genes in MPCs. We also examined the function of PGE(2)/EP4 signaling in the recovery of the HSPCs after myelosuppression. The administration of PGE(2) or an EP4 agonist facilitated the recovery of HSPCs from 5-fluorouracil (5-FU)-induced myelosuppression, indicating a role for PGE(2)/EP4 signaling in this process. Altogether, these data suggest that EP4 is a key receptor for PGE(2)-mediated direct and indirect regulation of HSPCs.

Enhanced Angpt1/Tie2 signaling affects the differentiation and long-term repopulation ability of hematopoietic stem cells.

Angiopoietin-1 (Angpt1) signaling via the Tie2 receptor regulates vascular and hematopoietic systems. To investigate the role of Angpt1-Tie2 signaling in hematopoiesis, we prepared conditionally inducible transgenic (Tg) mice expressing a genetically engineered Angpt1, cartridge oligomeric matrix protein (COMP)-Angpt1. The effects of COMP-Angpt1 overexpression in osteoblasts on hematopoiesis were then investigated by crossing COMP-Angpt1 Tg mice with Col1a1-Cre Tg mice. Interestingly, peripheral blood analyses showed that 4 week (wk)-old (but not 8 wk-old) Col1a1-Cre+/COMP-Angpt1+ mice had a lower percentage of circulating B cells and a higher percentage of myeloid cells than Col1a1-Cre-/COMP-Angpt1+ (control) mice. Although there were no significant differences in the immunophenotypic hematopoietic stem and progenitor cell (HSPC) populations between Col1a1-Cre+/COMP-Angpt1+ and control mice, lineage(-)Sca-1(+)c-Kit(+) (LSK) cells isolated from 8 wk-old Col1a1-Cre+/COMP-Angpt1+ mice showed better long-term bone marrow reconstitution ability. These data indicate that Angpt1-Tie2 signaling affects the differentiation capacity of hematopoietic lineages during development and increases the stem cell activity of HSCs.

Dosimetric comparison of rectal dose-reductive techniques in intra-cavitary brachytherapy for cervical cancer: A retrospective analysis.

Purpose: Rectal complications in radiotherapy for cervical cancer can highly affect quality of life and correlate with rectal dose. Vaginal gauze packing (VP) and rectal retraction (RR) are widely used for rectal dose reduction in high-dose-rate brachytherapy. We aimed to perform a dosimetric comparison of these two methods for three-dimensional image-guided adaptive brachytherapy. Material and methods: We retrospectively examined 50 patients with cervical cancer treated with definitive radiotherapy, including intra-cavitary brachytherapy, performed with VP and RR. We extracted two fractions for each patient: one fraction with VP and the next fraction with RR, and then compared dose-volume parameters. In total, 50 fractions each were analyzed in VP and RR groups. Dose to 90% (D90) of high-risk clinical target volume (HR-CTV), and minimum dose to most exposed 2.0 cm3 of other organs at risk (D2cm3) for the rectum and bladder were determined from planning computed tomography. Results: There were no significant differences between VP and RR in D90 of HR-CTV (mean: 7.479 Gy and 7.652 Gy, respectively, p = 0.172). The D2cm3 values for the rectum (mean: 4.234 Gy vs. 4.627 Gy, p = 0.008) and bladder (mean: 5.959 Gy vs. 6.690 Gy, p < 0.001) were significantly lower with VP compared with RR. Conclusions: VP reduced the dose to the rectum and bladder when compared with RR without impairing the dose to CTV.

N-cadherin+ HSCs in fetal liver exhibit higher long-term bone marrow reconstitution activity than N-cadherin- HSCs.

Adult hematopoietic stem cells (HSCs) are maintained in a microenvironment known as the stem cell niche. The regulation of HSCs in fetal liver (FL) and their niche, however, remains to be elucidated. In this study, we investigated the role of N-cadherin (N-cad) in the maintenance of HSCs during FL hematopoiesis. By using anti-N-cad antibodies (Abs) produced by our laboratory, we detected high N-cad expression in embryonic day 12.5 (E12.5) mouse FL HSCs, but not in E15.5 and E18.5 FL. Immunofluorescence staining revealed that N-cad(+)c-Kit(+) and N-cad(+) endothelial protein C receptor (EPCR)(+) HSCs co-localized with Lyve-1(+) sinusoidal endothelial cells (ECs) in E12.5 FL and that some of these cells also expressed N-cad. However, N-cad(+) HSCs were also observed to detach from the perisinusoidal niche at E15.5 and E18.5, concomitant with a down-regulation of N-cad and an up-regulation of E-cadherin (E-cad) in hepatic cells. Moreover, EPCR(+) long-term (LT)-HSCs were enriched in the N-cad(+)Lin(-)Sca-1(+)c-Kit(+) (LSK) fraction in E12.5 FL, but not in E15.5 or E18.5 FL. In a long-term reconstitution (LTR) activity assay, higher engraftment associated with N-cad(+) LSK cells versus N-cad(-) LSK cells in E12.5 FL when transplanted into lethally irradiated recipient mice. However, the higher engraftment of N-cad(+) LSK cells decreased subsequently in E15.5 and E18.5 FL. It is possible that N-cad expression conferred higher LTR activity to HSCs by facilitating interactions with the perisinusoidal niche, especially at E12.5. The down-regulation of N-cad during FL hematopoiesis may help us better understand the regulation and mobility of HSCs before migration into BM.

Homogenization of a phase-separated droplet in a polymer mixture caused by the dielectric effect of a laser.

It is found that a micrometer-sized droplet in an aqueous solution of binary polymers [water/polyethylene-glycol (PEG)/dextran] disappears upon irradiation with a focused yttrium-aluminum-garnet laser. The interface of the dextran-rich droplet broadens and disappears, and it reappears upon turning the laser off, whereas the PEG-rich droplet shrinks and disappears. These phenomena are discussed in terms of the free energy by considering the laser-induced dielectric potential.

Folliculin Regulates Osteoclastogenesis Through Metabolic Regulation.

Osteoclast differentiation is a dynamic differentiation process, which is accompanied by dramatic changes in metabolic status as well as in gene expression. Recent findings have revealed an essential connection between metabolic reprogramming and dynamic gene expression changes during osteoclast differentiation. However, the upstream regulatory mechanisms that drive these metabolic changes in osteoclastogenesis remain to be elucidated. Here, we demonstrate that induced deletion of a tumor suppressor gene, Folliculin (Flcn), in mouse osteoclast precursors causes severe osteoporosis in 3 weeks through excess osteoclastogenesis. Flcn-deficient osteoclast precursors reveal cell autonomous accelerated osteoclastogenesis with increased sensitivity to receptor activator of NF-κB ligand (RANKL). We demonstrate that Flcn regulates oxidative phosphorylation and purine metabolism through suppression of nuclear localization of the transcription factor Tfe3, thereby inhibiting expression of its target gene Pgc1. Metabolome studies revealed that Flcn-deficient osteoclast precursors exhibit significant augmentation of oxidative phosphorylation and nucleotide production, resulting in an enhanced purinergic signaling loop that is composed of controlled ATP release and autocrine/paracrine purinergic receptor stimulation. Inhibition of this purinergic signaling loop efficiently blocks accelerated osteoclastogenesis in Flcn-deficient osteoclast precursors. Here, we demonstrate an essential and novel role of the Flcn-Tfe3-Pgc1 axis in osteoclastogenesis through the metabolic reprogramming of oxidative phosphorylation and purine metabolism. © 2018 The Authors Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).

The telomere binding protein Pot1 maintains haematopoietic stem cell activity with age.

Repeated cell divisions and aging impair stem cell function. However, the mechanisms by which this occurs are not fully understood. Here we show that protection of telomeres 1A (Pot1a), a component of the Shelterin complex that protects telomeres, improves haematopoietic stem cell (HSC) activity during aging. Pot1a is highly expressed in young HSCs, but declines with age. In mouse HSCs, Pot1a knockdown increases DNA damage response (DDR) and inhibits self-renewal. Conversely, Pot1a overexpression or treatment with POT1a protein prevents DDR, maintained self-renewal activity and rejuvenated aged HSCs upon ex vivo culture. Moreover, treatment of HSCs with exogenous Pot1a inhibits the production of reactive oxygen species, suggesting a non-telomeric role for Pot1a in HSC maintenance. Consistent with these results, treatment with exogenous human POT1 protein maintains human HSC activity in culture. Collectively, these results show that Pot1a/POT1 sustains HSC activity and can be used to expand HSC numbers ex vivo.Repeated cell divisions induce DNA damage in haematopoietic stem cells (HSC) and telomeres are sensitive to this damage. Here, the authors show in murine HSCs that the telomere binding protein POT1a inhibited the production of reactive oxygen species, and rejuvenated aged HSCs.

Role of N-cadherin in the regulation of hematopoietic stem cells in the bone marrow niche.

Cell-cell and cell-extracellular matrix interactions between hematopoietic stem cells (HSCs) and their niches are critical for the maintenance of stem cell properties. Here, it is demonstrated that a cell adhesion molecule, N-cadherin, is expressed in hematopoietic stem/progenitor cells (HSPCs) and plays a critical role in the regulation of HSPC engraftment. Furthermore, overexpression of N-cadherin in HSCs promoted quiescence and preserved HSC activity during serial bone marrow (BM) transplantation (BMT). Inhibition of N-cadherin by the transduction of N-cadherin short hairpin (sh) RNA (shN-cad) reduced the lodgment of donor HSCs to the endosteal surface, resulting in a significant reduction in long-term engraftment. shN-cad-transduced cells were maintained in the spleen for six months after BMT, indicating that N-cadherin expression in HSCs is specifically required in the BM. These findings suggest that N-cadherin-mediated cell adhesion is functionally essential for the regulation of HSPC activities in the BM niche.