SUMMARY The regulation and coordination of mitochondrial metabolism with hematopoietic stem cell (HSC) self-renewal and differentiation is not fully understood. Here we report that depletion of PTPMT1, a PTEN-like mitochondrial phosphatase, in inducible or hematopoietic-cell-specific knockout mice resulted in hematopoietic failure due to changes in the cell cycle and a block in the differentiation of HSCs. Surprisingly, the HSC pool was increased by ~40-fold in PTPMT1 knockout mice. Reintroduction of wild-type PTPMT1, but not catalytically deficient PTPMT1 or truncated PTPMT1 lacking mitochondrial localization, restored differentiation capabilities of PTPMT1 knockout HSCs. Further analyses demonstrated that PTPMT1 deficiency altered mitochondrial metabolism and that phosphatidylinositol phosphate substrates of PTPMT1 directly enhanced fatty-acid-induced activation of mitochondrial uncoupling protein 2. Intriguingly, depletion of PTPMT1 from myeloid, T lymphoid, or B lymphoid progenitors did not cause any defects in lineage-specific knockout mice. This study establishes a crucial role of PTPMT1 in the metabolic regulation of HSC function.
Germline activating mutations of the protein tyrosine phosphatase SHP2 (encoded by PTPN11), a positive regulator of the RAS signalling pathway1, are found in 50% of patients with Noonan syndrome2. These patients have an increased risk of developing leukaemia3, especially juvenile myelomonocytic leukaemia (JMML), a childhood myeloproliferative neoplasm (MPN). Previous studies have demonstrated that mutations in Ptpn11 induce a JMML-like MPN through cell-autonomous mechanisms that are dependent on Shp2 catalytic activity4–7. However, the effect of these mutations in the bone marrow microenvironment remains unclear. Here we report that Ptpn11 activating mutations in the mouse bone marrow microenvironment promote the development and progression of MPN through profound detrimental effects on haematopoietic stem cells (HSCs). Ptpn11 mutations in mesenchymal stem/progenitor cells and osteoprogenitors, but not in differentiated osteoblasts or endothelial cells, cause excessive production of the CC chemokine CCL3 (also known as MIP-1α), which recruits monocytes to the area in which HSCs also reside. Consequently, HSCs are hyperactivated by interleukin-1β and possibly other proinflammatory cytokines produced by monocytes, leading to exacerbated MPN and to donor-cell-derived MPN following stem cell transplantation. Remarkably, administration of CCL3 receptor antagonists effectively reverses MPN development induced by the Ptpn11-mutated bone marrow microenvironment. This study reveals the critical contribution of Ptpn11 mutations in the bone marrow microenvironment to leukaemogenesis and identifies CCL3 as a potential therapeutic target for controlling leukaemic progression in Noonan syndrome and for improving stem cell transplantation therapy in Noonan-syndrome-associated leukaemias.
The intracellular Ca 2+ ([Ca 2+ ] i ) level of skeletal muscles must be rapidly regulated during the excitation-contraction-relaxation process 1 . However, the signaling components involved in such rapid Ca 2+ movement are not fully understood. Here, we report that mice deficient in the novel phosphatidylinositol phosphate (PIP) phosphatase MIP displayed muscle weakness and fatigue. Muscles isolated from MIP −/− mice produced less contractile force, markedly prolonged relaxation, and exhibited exacerbated fatigue. Further analyses revealed that MIP deficiency resulted in spontaneous Ca 2+ leak from the internal store -the sarcoplasmic reticulum (SR). This was attributed to the decreased metabolism/dephosphorylation and the subsequent accumulation of MIP substrates, especially PI(3,5)P 2 and PI(3,4)P 2 . Furthermore, we found that PI(3,5)P 2 and PI(3,4)P 2 bound to and directly activated the Ca 2+ release channel/ryanodine receptor (RyR1) of the SR. These studies provide the first evidence that finely controlled PIP levels in muscle cells are essential for maintaining Ca 2+ homeostasis and muscle performance.During our systematic genome-wide survey for tyrosine/dual specificity phosphatases (unpublished work), we discovered a novel phosphatase by hidden Markov database mining using the conserved catalytic motif ([V/I][V/I]HCXXGXXR[T/S]) as the bait sequence. Both human (BC035690) and mouse (BC018294) homologies were identified. They share 90% identity in amino acid sequences ( Supplementary Information, Fig. S1). Northern blotting analyses illustrated that this phosphatase was predominantly expressed in skeletal muscle and heart (Fig. 1a). Immunostaining indicates that it is primarily localized in the cytoplasm (data not shown). To verify its phosphatase property, we generated a GST fusion protein and tested its catalytic activity using pNPP (p-Nitrophenyl Phosphate), a widely used non-specific 7Correspondence should be addressed to: C.K.Q. (e-mail: E-mail: cxq6@case.edu). 6 These authors contributed equally to this work. AUTHOR CONTRIBUTIONSJ.S., W.M. Y., M.B., J.A.S., and C.S. conducted the research and summarized the data. C.K.Q., M.B., H.H.V., T.M.N., and C.G. designed the experiments and wrote the manuscript. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests. (Fig. 1b). Instead, it dephosphorylated a variety of PIPs, especially PI(3,5) P 2 (Fig. 1c), similar to PTEN and myotubularin and myopathy related (MTMR) phosphatases that also favor PIPs as substrates despite containing tyrosine phosphatase domains 2 . As this new phosphatase is mainly expressed in skeletal muscle and heart, we named it MIP (musclespecific inositol phosphatase). While our gene knockout work on MIP was ongoing, the Mustalin group also identified this phosphatase (FLJ20133) in their comprehensive collection of tyrosine phosphatases from the human genome and listed it as the 14 th member of the MTMR family (MTMR14) based on the homology of its catalytic motif to myotubularin 3 . More recently, ina...
The tissue inhibitors of metalloproteinases (TIMPs) regulate matrix metalloproteinase activity required for cell migration/invasion associated with cancer progression and angiogenesis. TIMPs also modulate cell proliferation in vitro and angiogenesis in vivo independent of their matrix metalloproteinase inhibitory activity. Here, we show that TIMP-2 mediates G 1 growth arrest in human endothelial cells through de novo synthesis of the cyclin-dependent kinase inhibitor p27 Kip1 . TIMP-2-mediated inhibition of Cdk4 and Cdk2 activity is associated with increased binding of p27Kip1 to these complexes in vivo. Protein-tyrosine phosphatase inhibitors or expression of a dominant negative Shp-1 mutant ablates TIMP-2 induction of p27 Kip1 . Finally, angiogenic responses to fibroblast growth factor-2 and vascular endothelial growth factor-A in "motheaten viable" Shp-1-deficient mice are resistant to TIMP-2 inhibition, demonstrating that Shp-1 is an important negative regulator of angiogenesis in vivo.Angiogenesis, the formation of new blood vessels from pre-existing vessels, accompanies a variety of pathologic responses in the adult, such as wound healing, tumor growth, cancer progression, and many chronic inflammatory diseases (1). Angiogenesis requires the dissolution of existing extracellular matrix and formation of new extracellular matrix, in particular the subendothelial basement membrane (2). The matrix metalloproteinases (MMPs) 2 have been demonstrated to play a pivotal role in angiogenesis through altering biological functions of extracellular matrix macromolecules by selectively degrading and/or releasing matrix-or membrane-anchored growth factors (3). Endogenous protease inhibitors, such as the tissue inhibitors of metalloproteinases (TIMPs), regulate the activities of these proteinases. TIMPs can suppress cell proliferation and invasion and reduce metastasis formation through inhibition of MMP activity and prevention of extracellular matrix turnover (4). However, recent studies suggest that TIMPs also directly modulate cell growth and migration via MMP-independent mechanisms (5-11).TIMPs 1-3 all inhibit angiogenesis; however, the mechanisms of these effects appear to be specific for each member of the TIMP family. TIMP-1 blocks tumor-associated angiogenesis via a mechanism involving inhibition of MMP-dependent endothelial cell migration (5, 12). However, a recent report suggests that TIMP-1 may also inhibit endothelial cell migration by an MMP-independent mechanism as well (13). In contrast, TIMP-3 prevents vascular endothelial growth factor-A (VEGF-A)-induced angiogenesis by direct antagonism of binding of this growth factor to its cognate receptor, . Previous studies have demonstrated that TIMP-2 inhibits the proliferation of endothelial cells, fibroblasts, and carcinoma cell lines in response to stimulation with mitogenic growth factors such as fibroblast growth factor-2 (FGF-2), platelet-derived growth factor (PDGF), or epidermal growth factor (EGF) (6, 9, 10). TIMP-2 binds to the surface of human micro...
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