Cosegregation of novel mitochondrial 16S rRNA gene mutations with the age-associated T414G variant in human cybrids

Ever increasing evidence has been provided on the accumulation of mutations in the mitochondrial DNA (mtDNA) during the aging process. However, the lack of direct functional consequences of the mutant mtDNA load on the mitochondria-dependent cell metabolism has raised many questions on the physiological importance of the age-related mtDNA variations. In the present work, we have analyzed the bioenergetic properties associated with the age-related T414G mutation of the mtDNA control region in transmitochondrial cybrids. The results show that the T414G mutation does not cause per se any detectable bioenergetic change. Moreover, three mtDNA mutations clustered in the 16S ribosomal RNA gene cosegregated together with the T414G in the same cybrid cell line. Two of them, namely T1843C and A1940G, are novel and associate with a negative bioenergetic phenotype. The results are discussed in the more general context of the complex heterogeneity and the dramatic instability of the mitochondrial genome during cell culture of transmitochondrial cybrids

A HGF/cMET Autocrine Loop Is Operative in Multiple Myeloma Bone Marrow Endothelial Cells and May Represent a Novel Therapeutic Target

Purpose: The aim of this study was to investigate the angiogenic role of the hepatocyte growth factor (HGF)/cMET pathway and its inhibition in bone marrow endothelial cells (EC) from patients with multiple myeloma versus from patients with monoclonal gammopathy of undetermined significance (MGUS) or benign anemia (control group). Experimental Design: The HGF/cMET pathway was evaluated in ECs from patients with multiple myeloma (multiple myeloma ECs) at diagnosis, at relapse after bortezomib- or lenalidomide-based therapies, or on refractory phase to these drugs; in ECs from patients with MGUS (MGECs); and in those patients from the control group. The effects of a selective cMET tyrosine kinase inhibitor (SU11274) on multiple myeloma ECs' angiogenic activities were studied in vitro and in vivo. Results: Multiple myeloma ECs express more HGF, cMET, and activated cMET (phospho (p)-cMET) at both RNAand protein levels versus MGECs and control ECs. Multiple myeloma ECs are able to maintain the HGF/cMET pathway activation in absence of external stimulation, whereas treatment with anti-HGF and anti-cMET neutralizing antibodies (Ab) is able to inhibit cMET activation. The cMET pathway regulates several multiple myeloma EC activities, including chemotaxis, motility, adhesion, spreading, and whole angiogenesis. Its inhibition by SU11274 impairs these activities in a statistically significant fashion when combined with bortezomib or lenalidomide, both in vitro and in vivo. Conclusions: An autocrine HGF/cMET loop sustains multiple myeloma angiogenesis and represents an appealing new target to potentiate the antiangiogenic management of patients with multiple myeloma

Hepatic-specific activation of peroxisome proliferator-activated receptor γ coactivator-1β protects against steatohepatitis

Development of hepatic steatosis and its progression to steatohepatitis may be the consequence of dysfunction of several metabolic pathways, such as triglyceride synthesis, very low-density lipoprotein (VLDL) secretion, and fatty acid β-oxidation. Peroxisome proliferator-activated receptor γ coactivator-1β (PGC-1β) is a master regulator of mitochondrial biogenesis and oxidative metabolism, lipogenesis, and triglyceride (TG) secretion. Here we generated a novel mouse model with constitutive hepatic activation of PGC-1β and studied the role of this transcriptional coactivator in dietary-induced steatosis and steatohepatitis. Selective activation of PGC-1β within hepatocytes is able to protect the liver from lipid overload and from progression to fibrosis. The protective function exerted by PGC-1β is due to its ability to induce mitochondrial oxidative phosphorylation, fatty acid β-oxidation, and citrate cycle, as well as to decrease oxidative stress and promote TG secretion in the blood stream. These findings bolster the concept that a combined hepatic specific action of PGC-1β on lipid synthesis and secretion, as well as on mitochondrial biogenesis and function, could protect against steatohepatitis

PGC-1β promotes enterocyte lifespan and tumorigenesis in the intestine

The mucosa of the small intestine is renewed completely every 3–5 d throughout the entire lifetime by small populations of adult stem cells that are believed to reside in the bottom of the crypts and to migrate and differentiate into all the different populations of intestinal cells. When the cells reach the apex of the villi and are fully differentiated, they undergo cell death and are shed into the lumen. Reactive oxygen species (ROS) production is proportional to the electron transfer activity of the mitochondrial respiration chain. ROS homeostasis is maintained to control cell death and is finely tuned by an inducible antioxidant program. Here we show that peroxisome proliferator-activated receptor-γ coactivator-1β (PGC-1β) is highly expressed in the intestinal epithelium and possesses dual activity, stimulating mitochondrial biogenesis and oxygen consumption while inducing antioxidant enzymes. To study the role of PGC-1β gain and loss of function in the gut, we generated both intestinal-specific PGC-1β transgenic and PGC-1β knockout mice. Mice overexpressing PGC-1β present a peculiar intestinal morphology with very long villi resulting from increased enterocyte lifespan and also demonstrate greater tumor susceptibility, with increased tumor number and size when exposed to carcinogens. PGC-1β knockout mice are protected from carcinogenesis. We show that PGC-1β triggers mitochondrial respiration while protecting enterocytes from ROS-driven macromolecule damage and consequent apoptosis in both normal and dysplastic mucosa. Therefore, PGC-1β in the gut acts as an adaptive self-point regulator, capable of providing a balance between enhanced mitochondrial activity and protection from increased ROS production

Mitochondrial dysregulation and oxidative stress in patients with chronic kidney disease

BACKGROUND: Chronic renal disease (CKD) is characterized by complex changes in cell metabolism leading to an increased production of oxygen radicals, that, in turn has been suggested to play a key role in numerous clinical complications of this pathological condition. Several reports have focused on the identification of biological elements involved in the development of systemic biochemical alterations in CKD, but this abundant literature results fragmented and not exhaustive. RESULTS: To better define the cellular machinery associated to this condition, we employed a high-throughput genomic approach based on a whole transcriptomic analysis associated with classical molecular methodologies. The genomic screening of peripheral blood mononuclear cells revealed that 44 genes were up-regulated in both CKD patients in conservative treatment (CKD, n = 9) and hemodialysis (HD, n = 17) compared to healthy subjects (HS, n = 8) (p < 0.001, FDR = 1%). Functional analysis demonstrated that 11/44 genes were involved in the oxidative phosphorylation system. Western blotting for COXI and COXIV, key constituents of the complex IV of oxidative phosphorylation system, performed on an independent testing-group (12 healthy subjects, 10 CKD and 14 HD) confirmed an higher synthesis of these subunits in CKD/HD patients compared to the control group. Only for COXI, the comparison between CKD and healthy subjects reached the statistical significance. However, complex IV activity was significantly reduced in CKD/HD patients compared to healthy subjects (p < 0.01). Finally, CKD/HD patients presented higher reactive oxygen species and 8-hydroxydeoxyguanosine levels compared to controls. CONCLUSION: Taken together these results suggest, for the first time, that CKD/HD patients may have an impaired mitochondrial respiratory system and this condition may be both the consequence and the cause of an enhanced oxidative stress

Characterization of ρ⁰ cells.

<p>A Multiplex PCR analysis of mitochondrial DNA. The nuclear and mitochondrial genes (18S rDNA: 229 bp and D-Loop: 436 bp, respectively) were coamplified and visualized by gel electrophoresis. Agarose gel (1.5%), lane 1 and 6: GeneRuler™ 100 bp plus DNA Ladder, lane 2: PC-3 wild type, lane 3: PC-3 ρ⁰ EtBr, lane 4: PC-3 ρ⁰ 9B4, lane 5: no template. <b>B</b> PCR analysis of EcoRI gene sequence in PC-3 ρ⁰ 9B4 cells. Different EcoRI gene sequences were amplified by PCR from PC-3 ρ⁰ 9B4 genomic DNA utilizing the primer pairs listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073207#pone-0073207-t002" target="_blank">Table 2</a>. Agarose gel (1.5%), lane 1 and 17: GeneRuler™ 100 bp Plus DNA Ladder, lane 2–4: PC-3 ρ⁰ 9B4, vector DNA (encoding mitochondrial targeted restriction endonuclease, 500 pg) and no template, primer pair A, lane 5–7: primer pair B, lane 8–10: primer pair C, lane 11–13: primer pair D, lane 14–16: primer pair E. <b>C</b> Cell growth analysis of PC-3 cells. Calculation of doubling time of PC-3 cells in media with uridine (dark grey bars) and without uridine (light grey bars). Total cell number was measured every 24 h over a period of six days and cell doubling was estimated using exponential regression. The presented data are means ± SD from four independent experiments. *P<0.05, **P<0.01, ***P<0.001.</p

Primer combinations for amplification of EcoRI gene sequence sections.

<p>Primer combinations for amplification of EcoRI gene sequence sections.</p

Relative quantification of mtDNA and cell growth analysis of PC-3 fusion cells.

<p>A Analysis of mtDNA relative to nuclear DNA over a period of 18 weeks after cell fusion of PC-3 ρ⁰ cells and cytoplasts of PC-3 MTS-DsRed (F 1–3, solid lines) or PC-3 MTS-EGFP ρ⁰ cells and cytoplasts of PC-3 MTS-DsRed (F A) and cytoplasts of 143B.TK^- MTS-DsRed (F B) (dashed lines) using a primer set for amplification of nuclear (18S rDNA) and mitochondrial (ND5 gene) DNA. Data were normalized to measurements of untreated wild type cells (dotted line). Data represent means ± SD obtained in three determinations. <b>B</b> Calculation of doubling time of PC-3 cells in media with uridine (dark grey bars) and without uridine (light grey bars). Total cell number was measured every 24 h over a period of six days and cell doubling was estimated using exponential regression. The presented data are means ± SD obtained in four experiments. Significance levels between wild type and the different fusion cell lines or if designated between two fusion cell lines were indicated. *P<0.05, **P<0.01, ***P<0.001. <i>B Inset</i> Comparison of relative mtDNA content and cell doubling time. MtDNA content and cell doubling time of PC-3 fusion cells cultured without uridine (close symbols) were normalized to wild type cells and a linear regression was performed (solid line).</p

Polarographic respiration measurements of PC-3 fusion cells.

<p><b>A</b> Endogenous oxygen consumption rate of intact PC-3 fusion cells was measured in TD buffer and normalized to cell number of the sample. <b>B</b> Relative oxygen consumption rates of PC-3 fusion cells were measured in TD buffer in absence and presence of the uncoupler DNP (dark grey bars). COX capacity of PC-3 fusion cells was measured as relative uncoupled respiration rate at 0.4 mM TMPD in antimycin-treated cells (light grey bars). The data represent means ± SD from 4–6 independent experiments. *P<0.05, **P<0.01, ***P<0.001.</p