Parathyroid hormone regulates fates of murine osteoblast precursors in vivo

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Abstract

Teriparatide, a recombinant form of parathyroid hormone (PTH), is the only approved treatment for osteoporosis that increases the rate of bone formation. Teriparatide increases osteoblast numbers by suppressing osteoblast apoptosis and activating bone-lining cells. No direct evidence for teriparatide’s actions on early cells of the osteoblast lineage has been demonstrated. Here, we have employed a lineage-tracing strategy that uses a tamoxifen-dependent, promoter-driven cre to mark early cells of the osteoblast lineage in adult mice. We show that teriparatide increases the numbers of osteoblast precursors and drives their differentiation into mature osteoblasts. Unexpectedly, following withdrawal of teriparatide therapy, bone marrow adipocytes increased dramatically in number. Some of these adipocytes derived from cells marked by Sox9-cre expression weeks earlier. Continued therapy with teriparatide prevented the appearance of adipocytes. Selective, inducible deletion of the PTH receptor in Sox9-cre cells demonstrated that PTH receptor expression is required for teriparatide-mediated increases in early osteoblast precursors. The increase in early precursors after teriparatide administration was associated with robust suppression of precursor apoptosis without affecting their rate of proliferation. Thus, teriparatide increases the numbers of early cells of the osteoblast lineage, hastens their differentiation into osteoblasts, and suppresses their differentiation into adipocytes in vivo.

Authors

Deepak H. Balani, Noriaki Ono, Henry M. Kronenberg

Activin-A enhances mTOR signaling to promote aberrant chondrogenesis in fibrodysplasia ossificans progressiva

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Abstract

Fibrodysplasia ossificans progressiva (FOP) is a rare and intractable disease characterized by extraskeletal bone formation through endochondral ossification. Patients with FOP harbor point mutations in ACVR1, a type I receptor for BMPs. Although mutated ACVR1 (FOP-ACVR1) has been shown to render hyperactivity in BMP signaling, we and others have uncovered a mechanism by which FOP-ACVR1 mistransduces BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling. Although Activin-A evokes enhanced chondrogenesis in vitro and heterotopic ossification (HO) in vivo, the underlying mechanisms have yet to be revealed. To this end, we developed a high-throughput screening (HTS) system using FOP patient–derived induced pluripotent stem cells (FOP-iPSCs) to identify pivotal pathways in enhanced chondrogenesis that are initiated by Activin-A. In a screen of 6,809 small-molecule compounds, we identified mTOR signaling as a critical pathway for the aberrant chondrogenesis of mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs). Two different HO mouse models, an FOP model mouse expressing FOP-ACVR1 and an FOP-iPSC–based HO model mouse, revealed critical roles for mTOR signaling in vivo. Moreover, we identified ENPP2, an enzyme that generates lysophosphatidic acid, as a linker of FOP-ACVR1 and mTOR signaling in chondrogenesis. These results uncovered the crucial role of the Activin-A/FOP-ACVR1/ENPP2/mTOR axis in FOP pathogenesis.

Authors

Kyosuke Hino, Kazuhiko Horigome, Megumi Nishio, Shingo Komura, Sanae Nagata, Chengzhu Zhao, Yonghui Jin, Koichi Kawakami, Yasuhiro Yamada, Akira Ohta, Junya Toguchida, Makoto Ikeya

Targeting NEK2 attenuates glioblastoma growth and radioresistance by destabilizing histone methyltransferase EZH2

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Abstract

Accumulating evidence suggests that glioma stem cells (GSCs) are important therapeutic targets in glioblastoma (GBM). In this study, we identified NIMA-related kinase 2 (NEK2) as a functional binding protein of enhancer of zeste homolog 2 (EZH2) that plays a critical role in the posttranslational regulation of EZH2 protein in GSCs. NEK2 was among the most differentially expressed kinase-encoding genes in GSC-containing cultures (glioma spheres), and it was required for in vitro clonogenicity, in vivo tumor propagation, and radioresistance. Mechanistically, the formation of a protein complex comprising NEK2 and EZH2 in glioma spheres phosphorylated and then protected EZH2 from ubiquitination-dependent protein degradation in a NEK2 kinase activity–dependent manner. Clinically, NEK2 expression in patients with glioma was closely associated with EZH2 expression and correlated with a poor prognosis. NEK2 expression was also substantially elevated in recurrent tumors after therapeutic failure compared with primary untreated tumors in matched GBM patients. We designed a NEK2 kinase inhibitor, compound 3a (CMP3a), which efficiently attenuated GBM growth in a mouse model and exhibited a synergistic effect with radiotherapy. These data demonstrate a key role for NEK2 in maintaining GSCs in GBM by stabilizing the EZH2 protein and introduce the small-molecule inhibitor CMP3a as a potential therapeutic agent for GBM.

Authors

Jia Wang, Peng Cheng, Marat S. Pavlyukov, Hai Yu, Zhuo Zhang, Sung-Hak Kim, Mutsuko Minata, Ahmed Mohyeldin, Wanfu Xie, Dongquan Chen, Violaine Goidts, Brendan Frett, Wenhao Hu, Hongyu Li, Yong Jae Shin, Yeri Lee, Do-Hyun Nam, Harley I. Kornblum, Maode Wang, Ichiro Nakano

mRNA-mediated glycoengineering ameliorates deficient homing of human stem cell–derived hematopoietic progenitors

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Abstract

Generation of functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) has been a long-sought-after goal for use in hematopoietic cell production, disease modeling, and eventually transplantation medicine. Homing of HSPCs from bloodstream to bone marrow (BM) is an important aspect of HSPC biology that has remained unaddressed in efforts to derive functional HSPCs from human PSCs. We have therefore examined the BM homing properties of human induced pluripotent stem cell–derived HSPCs (hiPS-HSPCs). We found that they express molecular effectors of BM extravasation, such as the chemokine receptor CXCR4 and the integrin dimer VLA-4, but lack expression of E-selectin ligands that program HSPC trafficking to BM. To overcome this deficiency, we expressed human fucosyltransferase 6 using modified mRNA. Expression of fucosyltransferase 6 resulted in marked increases in levels of cell surface E-selectin ligands. The glycoengineered cells exhibited enhanced tethering and rolling interactions on E-selectin–bearing endothelium under flow conditions in vitro as well as increased BM trafficking and extravasation when transplanted into mice. However, glycoengineered hiPS-HSPCs did not engraft long-term, indicating that additional functional deficiencies exist in these cells. Our results suggest that strategies toward increasing E-selectin ligand expression could be applicable as part of a multifaceted approach to optimize the production of HSPCs from human PSCs.

Authors

Jungmin Lee, Brad Dykstra, Joel A. Spencer, Laurie L. Kenney, Dale L. Greiner, Leonard D. Shultz, Michael A. Brehm, Charles P. Lin, Robert Sackstein, Derrick J. Rossi

Long-lived keratin 15⁺ esophageal progenitor cells contribute to homeostasis and regeneration

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Abstract

The esophageal lumen is lined by a stratified squamous epithelium comprised of proliferative basal cells that differentiate while migrating toward the luminal surface and eventually desquamate. Rapid epithelial renewal occurs, but the specific cell of origin that supports this high proliferative demand remains unknown. Herein, we have described a long-lived progenitor cell population in the mouse esophageal epithelium that is characterized by expression of keratin 15 (Krt15). Genetic in vivo lineage tracing revealed that the Krt15 promoter marks a long-lived basal cell population able to self-renew, proliferate, and generate differentiated cells, consistent with a progenitor/stem cell population. Transcriptional profiling demonstrated that Krt15⁺ basal cells are molecularly distinct from Krt15^– basal cells. Depletion of Krt15-derived cells resulted in decreased proliferation, thereby leading to atrophy of the esophageal epithelium. Further, Krt15⁺ cells were radioresistant and contributed to esophageal epithelial regeneration following radiation-induced injury. These results establish the presence of a long-lived and indispensable Krt15⁺ progenitor cell population that provides additional perspective on esophageal epithelial biology and the widely prevalent diseases that afflict this epithelium.

Authors

Véronique Giroux, Ashley A. Lento, Mirazul Islam, Jason R. Pitarresi, Akriti Kharbanda, Kathryn E. Hamilton, Kelly A. Whelan, Apple Long, Ben Rhoades, Qiaosi Tang, Hiroshi Nakagawa, Christopher J. Lengner, Adam J. Bass, E. Paul Wileyto, Andres J. Klein-Szanto, Timothy C. Wang, Anil K. Rustgi

Prospective isolation of NKX2-1–expressing human lung progenitors derived from pluripotent stem cells

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Abstract

It has been postulated that during human fetal development, all cells of the lung epithelium derive from embryonic, endodermal, NK2 homeobox 1–expressing (NKX2-1⁺) precursor cells. However, this hypothesis has not been formally tested owing to an inability to purify or track these progenitors for detailed characterization. Here we have engineered and developmentally differentiated NKX2-1^GFP reporter pluripotent stem cells (PSCs) in vitro to generate and isolate human primordial lung progenitors that express NKX2-1 but are initially devoid of differentiated lung lineage markers. After sorting to purity, these primordial lung progenitors exhibited lung epithelial maturation. In the absence of mesenchymal coculture support, this NKX2-1⁺ population was able to generate epithelial-only spheroids in defined 3D cultures. Alternatively, when recombined with fetal mouse lung mesenchyme, the cells recapitulated epithelial-mesenchymal developing lung interactions. We imaged these progenitors in real time and performed time-series global transcriptomic profiling and single-cell RNA sequencing as they moved through the earliest moments of lung lineage specification. The profiles indicated that evolutionarily conserved, stage-dependent gene signatures of early lung development are expressed in primordial human lung progenitors and revealed a CD47^hiCD26^lo cell surface phenotype that allows their prospective isolation from untargeted, patient-specific PSCs for further in vitro differentiation and future applications in regenerative medicine.

Authors

Finn Hawkins, Philipp Kramer, Anjali Jacob, Ian Driver, Dylan C. Thomas, Katherine B. McCauley, Nicholas Skvir, Ana M. Crane, Anita A. Kurmann, Anthony N. Hollenberg, Sinead Nguyen, Brandon G. Wong, Ahmad S. Khalil, Sarah X.L. Huang, Susan Guttentag, Jason R. Rock, John M. Shannon, Brian R. Davis, Darrell N. Kotton

Disruption of lineage specification in adult pulmonary mesenchymal progenitor cells promotes microvascular dysfunction

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Abstract

Pulmonary vascular disease is characterized by remodeling and loss of microvessels and is typically attributed to pathological responses in vascular endothelium or abnormal smooth muscle cell phenotypes. We have challenged this understanding by defining an adult pulmonary mesenchymal progenitor cell (MPC) that regulates both microvascular function and angiogenesis. The current understanding of adult MPCs and their roles in homeostasis versus disease has been limited by a lack of genetic markers with which to lineage label multipotent mesenchyme and trace the differentiation of these MPCs into vascular lineages. Here, we have shown that lineage-labeled lung MPCs expressing the ATP-binding cassette protein ABCG2 (ABCG2⁺) are pericyte progenitors that participate in microvascular homeostasis as well as adaptive angiogenesis. Activation of Wnt/β-catenin signaling, either autonomously or downstream of decreased BMP receptor signaling, enhanced ABCG2⁺ MPC proliferation but suppressed MPC differentiation into a functional pericyte lineage. Thus, enhanced Wnt/β-catenin signaling in ABCG2⁺ MPCs drives a phenotype of persistent microvascular dysfunction, abnormal angiogenesis, and subsequent exacerbation of bleomycin-induced fibrosis. ABCG2⁺ MPCs may, therefore, account in part for the aberrant microvessel function and remodeling that are associated with chronic lung diseases.

Authors

Christa F. Gaskill, Erica J. Carrier, Jonathan A. Kropski, Nathaniel C. Bloodworth, Swapna Menon, Robert F. Foronjy, M. Mark Taketo, Charles C. Hong, Eric D. Austin, James D. West, Anna L. Means, James E. Loyd, W. David Merryman, Anna R. Hemnes, Stijn De Langhe, Timothy S. Blackwell, Dwight J. Klemm, Susan M. Majka

Disruption of spatiotemporal hypoxic signaling causes congenital heart disease in mice

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Abstract

Congenital heart disease (CHD) represents the most prevalent inborn anomaly. Only a minority of CHD cases are attributed to genetic causes, suggesting a major role of environmental factors. Nonphysiological hypoxia during early pregnancy induces CHD, but the underlying reasons are unknown. Here, we have demonstrated that cells in the mouse heart tube are hypoxic, while cardiac progenitor cells (CPCs) expressing islet 1 (ISL1) in the secondary heart field (SHF) are normoxic. In ISL1⁺ CPCs, induction of hypoxic responses caused CHD by repressing Isl1 and activating NK2 homeobox 5 (Nkx2.5), resulting in decreased cell proliferation and enhanced cardiomyocyte specification. We found that HIF1α formed a complex with the Notch effector hes family bHLH transcription factor 1 (HES1) and the protein deacetylase sirtuin 1 (SIRT1) at the Isl1 gene. This complex repressed Isl1 in the hypoxic heart tube or following induction of ectopic hypoxic responses. Subsequently, reduced Isl1 expression abrogated ISL1-dependent recruitment of histone deacetylases HDAC1/5, inhibiting Nkx2.5 expression. Inactivation of Sirt1 in ISL1⁺ CPCs blocked Isl1 suppression via the HIF1α/HES1/SIRT1 complex and prevented CHDs induced by pathological hypoxia. Our results indicate that spatial differences in oxygenation of the developing heart serve as signals to control CPC expansion and cardiac morphogenesis. We propose that physiological hypoxia coordinates homeostasis of CPCs, providing mechanistic explanations for some nongenetic causes of CHD.

Authors

Xuejun Yuan, Hui Qi, Xiang Li, Fan Wu, Jian Fang, Eva Bober, Gergana Dobreva, Yonggang Zhou, Thomas Braun

Amino acid–insensitive mTORC1 regulation enables nutritional stress resilience in hematopoietic stem cells

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Abstract

The mTOR pathway is a critical determinant of cell persistence and growth wherein mTOR complex 1 (mTORC1) mediates a balance between growth factor stimuli and nutrient availability. Amino acids or glucose facilitates mTORC1 activation by inducing RagA GTPase recruitment of mTORC1 to the lysosomal outer surface, enabling activation of mTOR by the Ras homolog Rheb. Thereby, RagA alters mTORC1-driven growth in times of nutrient abundance or scarcity. Here, we have evaluated differential nutrient-sensing dependence through RagA and mTORC1 in hematopoietic progenitors, which dynamically drive mature cell production, and hematopoietic stem cells (HSC), which provide a quiescent cellular reserve. In nutrient-abundant conditions, RagA-deficient HSC were functionally unimpaired and upregulated mTORC1 via nutrient-insensitive mechanisms. RagA was also dispensable for HSC function under nutritional stress conditions. Similarly, hyperactivation of RagA did not affect HSC function. In contrast, RagA deficiency markedly altered progenitor population function and mature cell output. Therefore, RagA is a molecular mechanism that distinguishes the functional attributes of reactive progenitors from a reserve stem cell pool. The indifference of HSC to nutrient sensing through RagA contributes to their molecular resilience to nutritional stress, a characteristic that is relevant to organismal viability in evolution and in modern HSC transplantation approaches.

Authors

Demetrios Kalaitzidis, Dongjun Lee, Alejo Efeyan, Youmna Kfoury, Naema Nayyar, David B. Sykes, Francois E. Mercier, Ani Papazian, Ninib Baryawno, Gabriel D. Victora, Donna Neuberg, David M. Sabatini, David T. Scadden

FOXP1 controls mesenchymal stem cell commitment and senescence during skeletal aging

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Abstract

A hallmark of aged mesenchymal stem/progenitor cells (MSCs) in bone marrow is the pivot of differentiation potency from osteoblast to adipocyte coupled with a decrease in self-renewal capacity. However, how these cellular events are orchestrated in the aging progress is not fully understood. In this study, we have used molecular and genetic approaches to investigate the role of forkhead box P1 (FOXP1) in transcriptional control of MSC senescence. In bone marrow MSCs, FOXP1 expression levels declined with age in an inverse manner with those of the senescence marker p16^INK4A. Conditional depletion of Foxp1 in bone marrow MSCs led to premature aging characteristics, including increased bone marrow adiposity, decreased bone mass, and impaired MSC self-renewal capacity in mice. At the molecular level, FOXP1 regulated cell-fate choice of MSCs through interactions with the CEBPβ/δ complex and recombination signal binding protein for immunoglobulin κ J region (RBPjκ), key modulators of adipogenesis and osteogenesis, respectively. Loss of p16^INK4A in Foxp1-deficient MSCs partially rescued the defects in replication capacity and bone mass accrual. Promoter occupancy analyses revealed that FOXP1 directly represses transcription of p16^INK4A. These results indicate that FOXP1 attenuates MSC senescence by orchestrating their cell-fate switch while maintaining their replicative capacity in a dose- and age-dependent manner.

Authors

Hanjun Li, Pei Liu, Shuqin Xu, Yinghua Li, Joseph D. Dekker, Baojie Li, Ying Fan, Zhenlin Zhang, Yang Hong, Gong Yang, Tingting Tang, Yongxin Ren, Haley O. Tucker, Zhengju Yao, Xizhi Guo