Massimiliano Monticone

CRTAP Is Required for Prolyl 3- Hydroxylation and Mutations Cause Recessive Osteogenesis Imperfecta

Prolyl hydroxylation is a critical posttranslational modification that affects structure, function, and turnover of target proteins. Prolyl 3-hydroxylation occurs at only one position in the triple-helical domain of fibrillar collagen chains, and its biological significance is unknown. CRTAP shares homology with a family of putative prolyl 3-hydroxylases (P3Hs), but it does not contain their common dioxygenase domain. Loss of Crtap in mice causes an osteochondrodysplasia characterized by severe osteoporosis and decreased osteoid production. CRTAP can form a complex with P3H1 and cyclophilin B (CYPB), and Crtap-/- bone and cartilage collagens show decreased prolyl 3-hydroxylation. Moreover, mutant collagen shows evidence of overmodification, and collagen fibrils in mutant skin have increased diameter consistent with altered fibrillogenesis. In humans, CRTAP mutations are associated with the clinical spectrum of recessive osteogenesis imperfecta, including the type II and VII forms. Hence, dysregulation of prolyl 3-hydroxylation is a mechanism for connective tissue disease.

Evidence for aerobic metabolism in retinal rod outer segment disks.

The disks of the vertebrate retinal rod Outer Segment (OS), devoid of mitochondria, are the site of visual transduction, a very energy demanding process. In a previous proteomic study we reported the expression of the respiratory chain complexes I-IV and the oxidative phosphorylation Complex V (F(1)F(0)-ATP synthase) in disks. In the present study, the functional localization of these proteins in disks was investigated by biochemical analyses, oxymetry, membrane potential measurements, and confocal laser scanning microscopy. Disk preparations, isolated by Ficoll flotation, were characterized for purity. An oxygen consumption, stimulated by NADH and Succinate and reverted by rotenone, antimycin A and KCN was measured in disks, either in coupled or uncoupled conditions. Rhodamine-123 fluorescence quenching kinetics showed the existence of a proton potential difference across the disk membranes. Citrate synthase activity was assayed and found enriched in disks with respect to ROS. ATP synthesis by disks (0.7 micromol ATP/min/mg), sensitive to the common mitochondrial ATP synthase inhibitors, would largely account for the rod ATP need in the light. Overall, data indicate that an oxidative phosphorylation occurs in rod OS, which do not contain mitochondria, thank to the presence of ectopically located mitochondrial proteins. These findings may provide important new insight into energy production in outer segments via aerobic metabolism and additional information about protein components in OS disk membranes.

cDNA cloning, characterization and chromosome mapping of Crtap encoding the mouse cartilage associated protein.

Recently we have isolated and characterized a cDNA coding for a novel developmentally regulated chick embryo protein, cartilage associated protein (CASP). Here we describe the isolation and characterization of the cDNAs coding for the mouse CASP. Comparison of the mammalian putative protein sequence with the chick sequence shows a very high identity overall (51%); in particular the chick protein is homologous to the half amino terminus of the mouse protein. Furthermore, the comparison of the CASP cDNA sequence with sequences of the genebank database confirms our hypothesis that the CASP genes belong to a novel family that also includes genes encoding for some nuclear antigens. In all mouse tissues examined three CASP mRNAs species are detected, whereas in chick tissues a single mRNA is present. Immunohistochemistry studies show that the protein is expressed in all mouse embryonic cartilages. The mouse cartilage associated protein gene (Crtap) was assigned to chromosome 9F3-F4 by fluorescence in situ hybridization.

Activation of nervous system development genes in bone marrow derived mesenchymal stem cells following spaceflight exposure

Stalled cell division in precursor bone cells and reduced osteoblast function are considered responsible for the microgravity‐induced bone loss observed during spaceflight. However, underlying molecular mechanisms remain unraveled. Having overcome technological difficulties associated with flying cells in a space mission, we present the first report on the behavior of the potentially osteogenic murine bone marrow stromal cells (BMSC) in a 3D culture system, flown inside the KUBIK aboard space mission ISS 12S (Soyuz TMA‐8 + Increment 13) from March 30 to April 8, 2006 (experiment “Stroma‐2”). Flight 1g control cultures were performed in a centrifuge located within the payload. Ground controls were maintained on Earth in another KUBIK payload and in Petri dishes. Half of the cultures were stimulated with osteo‐inductive medium. Differences in total RNA extracted suggested that cell proliferation was inhibited in flight samples. Affymetrix technology revealed that 1,599 genes changed expression after spaceflight exposure. A decreased expression of cell‐cycle genes confirmed the inhibition of cell proliferation in space. Unexpectedly, most of the modulated expression was found in genes related to various processes of neural development, neuron morphogenesis, transmission of nerve impulse and synapse, raising the question on the lineage restriction in BMSC. J. Cell. Biochem. 111: 442–452, 2010.

Gene expression profile of human bone marrow stromal cells determined by restriction fragment differential display analysis.

Using an in vitro osteogenic culture system, we carried out a restriction fragment differential display (RFDD‐PCR) to identify genes expressed by these cells in their undifferentiated stage and not expressed, or expressed at a lower level, in a closely related but distinct cell type: bone marrow stromal cells (BMSC)‐derived osteoblasts (BDO). Forty‐seven candidate regulated genes, selected by RFDD, were analyzed by RT‐PCR analysis in three cell clones and in primary cultures from seven different donors. A subset of three genes were confirmed as upregulated in BMSC relative to BDO in every primary culture and cloned population examined: βIG‐h3, IGFbp3, and LOXL2. Their differential expression was confirmed by Northern analysis and the corresponding proteins were detected by immunolocalization in BMSC.

Gene expression deregulation by KRAS G12D and G12V in a BRAF V600E context

BackgroundKRAS and BRAF mutations appear of relevance in the genesis and progression of several solid tumor types but the co-occurrence and interaction of these mutations have not yet been fully elucidated. Using a microsatellite stable (MSS) colorectal cancer (CRC) cell line (Colo741) having mutated BRAF and KRASWT, we also aimed to investigate the KRAS-BRAF interaction. Gene expression profiles for control KRASWT, KRASG 12Vand KRASG 12Dtransfected cells were obtained after cell clone selection and RT-PCR screening. Extensive qPCR was performed to confirm microarray data.ResultsWe found that the KRASG 12Vstate deregulated several genes associated to cell cycle, apoptosis and nitrogen metabolism. These findings indicated a reduced survival and proliferation with respect to the KRASWTstate. The KRASG 12Dstate was, instead, characterized by several other distinct functional changes as for example those related to chromatin organization and cell-cell adhesion without affecting apoptosis related genes.ConclusionThese data predict that the G12D mutation may be more likely selected in a BRAF mutated context. At the same time, the presence of the KRASG 12Vmutation in the cells escaping apoptosis and inducing angiogenesis via IL8 may confer a more aggressive phenotype. The present results get along with the observations that CRCs with G12V are associated with a worse prognosis with respect to the WT and G12D states and may help identifying novel CRC pathways and biomarkers of clinical relevance.

z-Leucinyl-leucinyl-norleucinal induces apoptosis of human glioblastoma tumor-initiating cells by proteasome inhibition and mitotic arrest response.

γ-secretase inhibitors have been proposed as drugs able to kill cancer cells by targeting the NOTCH pathway. Here, we investigated two of such inhibitors, the Benzyloxicarbonyl-Leu-Leu-Nle-CHO (LLNle) and the N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), to assess whether they were effective in killing human glioblastoma tumor–initiating cells (GBM TIC) in vitro. We found that only LLNle was able at the micromolar range to induce the death of GBM TICs by apoptosis. To determine the cellular processes that were activated in GBM TICs by treatment with LLNle, we analyzed the amount of the NOTCH intracellular domain and the gene expression profiles following treatment with LLNle, DAPT, and DMSO (vehicle). We found that LLNIe, beside inhibiting the generation of the NOTCH intracellular domain, also induces proteasome inhibition, proteolytic stress, and mitotic arrest in these cells by repressing genes required for DNA synthesis and mitotic progression and by activating genes acting as mitotic inhibitors. DNA content flow cytometry clearly showed that cells treated with LLNle undergo arrest in the G2-M phases of the cell cycle. We also found that DAPT and L-685,458, another selective Notch inhibitor, were unable to kill GBM TICs, whereas lactacystin, a pure proteasome inhibitor, was effective although at a much less extent than LLNle. These data show that LLNle kills GBM TIC cells by inhibiting the proteasome activity. We suggest that LLNle, being able to target two relevant pathways for GBM TIC survival, may have a potential therapeutic value that deserves further investigation in animal models. (Mol Cancer Res 2009;7(11):1822–34)

NAC, Tiron and Trolox Impair Survival of Cell Cultures Containing Glioblastoma Tumorigenic Initiating Cells by Inhibition of Cell Cycle Progression

Reactive oxygen species (ROS) are metabolism by-products that may act as signaling molecules to sustain tumor growth. Antioxidants have been used to impair cancer cell survival. Our goal was to determine the mechanisms involved in the response to antioxidants of a human cell culture (PT4) containing glioblastoma (GBM) tumorigenic initiating cells (TICs). ROS production in the absence or presence of N-acetyl-L-cysteine (NAC), tiron, and trolox was evaluated by flow cytometry (FCM). The effects of these antioxidants on cell survival and apoptosis were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) and FCM. The biological processes modulated by these drugs were determined by oligonucleotide microarray gene expression profiling. Our results showed that NAC, tiron and trolox impaired PT4 cell survival, had minor effects on ROS levels and caused wide deregulation of cell cycle genes. Furthermore, tiron and trolox caused inhibition of cell survival in two additional cell cultures containing TICs, FO-1 and MM1, established from a melanoma and a mesothelioma patient, respectively. NAC, instead, impaired survival of the MM1 cells but not of the FO-1 cells. However, when used in combination, NAC enhanced the inhibitory effect of PLX4032 (BRAF V600E inhibitor) and Gefitinib (EGFR inhibitor), on FO-1 and PT4 cell survival. Collectively, NAC, tiron and trolox modulated gene expression and impaired the growth of cultures containing TICs primarily by inhibiting cell cycle progression.

The Nuclear Genes Mtfr1 and Dufd1 Regulate Mitochondrial Dynamic and Cellular Respiration

Dufd1 (DUF729 domain containing 1) is related to Mtfr1 (mitochondrial fission regulator 1), a gene involved in the regulation of antioxidant activity in the mouse testis. The present study was undertaken to better understand their role in regulating mitochondrial architecture and function in the mouse. We show that Dufd1 is expressed as a 2 kb mRNA and has a more specific tissue pattern compared to Mtfr1, with highest level of expression in testes, lower level in spleen, and negligible levels in other organs and/or tissues. In the male gonad, Dufd1 mRNA expression increases during postnatal development, similarly to Mtfr1. In situ hybridization and real‐time PCR analyses show that Dufd1 is expressed in the seminiferous tubules by middle‐late pachytene spermatocytes and spermatids. In transfected cells, the Dufd1‐tagged protein is located in mitochondria, associated with the tips of mitochondrial tubules and to tubules constrictions, and induces mitochondrial fission although with a lesser efficiency than Mtfr1. We also found that both endogenous Dufd1 and Mtfr1 proteins are associated with membrane‐enriched subcellular fractions, including mitochondria. Inhibition of Mtfr1 and/or Dufd1 expression, in a testicular germ cells line, severely impairs O2 consumption and indicates that both genes are required for mitochondrial respiration. Accordingly, analysis of testes mitochondria from Mtfr1‐deficient mice reveals severely reduced O2 consumption and ATP synthesis compared to wt animals. These data show that, in murine testis, Dufd1 and Mtfr1 have redundant functions related to mitochondrial physiology and represent genes with a potential role in testicular function. J. Cell. Physiol. 225: 767–776, 2010.

Chondrocyte protein with a poly-proline region (CHPPR) is a novel mitochondrial protein and promotes mitochondrial fission

We have recently identified a chondrocyte protein with a poly‐proline region, referred to as CHPPR, and showed that this protein is expressed intracellularly in chick embryo chondrocytes. Conventional fluorescence and confocal localization of CHPPR shows that CHPPR is sorted to mitochondria. Furthermore, immunoelectron microscopy of CHPPR transfected cells demonstrates that this protein is mostly associated with the mitochondrial inner membranes. Careful analysis of CHPPR expressing cells reveals, instead of the regular mitochondrial tubular network, the presence of a number of small spheroid mitochondria. Here we show that the domain responsible for network–spheroid transition spans amino acid residues 182–309 including the poly‐proline region. Functional analyses of mitochondrial activity rule out the possibility of mitochondrial damage in CHPPR transfected cells. Since cartilage expresses high levels of CHPPR mRNA when compared to other tissues and because CHPPR is associated with late stages of chondrocyte differentiation, we have investigated mitochondrial morphology in hypertrophic chondrocytes by MitoTracker Orange labeling. Confocal microscopy shows that these cells have spheroid mitochondria. Our data demonstrate that CHPPR is able to promote mitochondrial fission with a sequence specific mechanism suggesting that this event may be relevant to late stage of chondrocyte differentiation.