Cell intrinsic and extrinsic mechanisms of stem cell aging depend on telomere status

The function of adult stem cells declines during aging and chronic diseases. An understanding of the molecular mechanisms underlying these processes will help to identify targets for future therapies in order to improve regenerative reserve and organ maintenance. Telomere shortening represents a cell intrinsic mechanism inducing DNA damage in aging cells. Current studies in telomerase knockout mice have shown that telomere dysfunction induces cell intrinsic checkpoints and environmental alteration that limit stem cell function. While these phenotypes differ from wild-type mice with long telomere reserves, they appear to be relevant for human aging, which is associated with an accumulation of telomere dysfunction and DNA damage.     

Cell metabolism in the regulation of programmed cell death.

Increased cellular metabolism and resistance to apoptosis are two hallmarks of cell transformation. Recent progress in the understanding of the role of mitochondria in controlling apoptosis has brought attention to the links between elements of the apoptotic machinery and cellular metabolism. Here, we review the coordinated effects of growth factor withdrawal on bioenergetics and programmed cell death, and discuss the metabolic consequences of genes that prevent apoptosis, including the BCL2 family of genes and AKT.     

RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism

We have generated RANK (receptor activator of NF-kappaB) nullizygous mice to determine the molecular genetic interactions between osteoprotegerin, osteoprotegerin ligand, and RANK during bone resorption and remodeling processes. RANK(-/-) mice lack osteoclasts and have a profound defect in bone resorption and remodeling and in the development of the cartilaginous growth plates of endochondral bone. The osteopetrosis observed in these mice can be reversed by transplantation of bone marrow from rag1(-/-) (recombinase activating gene 1) mice, indicating that RANK(-/-) mice have an intrinsic defect in osteoclast function. Calciotropic hormones and proresorptive cytokines that are known to induce bone resorption in mice and human were administered to RANK(-/-) mice without inducing hypercalcemia, although tumor necrosis factor alpha treatment leads to the rare appearance of osteoclast-like cells near the site of injection. Osteoclastogenesis can be initiated in RANK(-/-) mice by transfer of the RANK cDNA back into hematopoietic precursors, suggesting a means to critically evaluate RANK structural features required for bone resorption. Together these data indicate that RANK is the intrinsic cell surface determinant that mediates osteoprotegerin ligand effects on bone resorption and remodeling as well as the physiological and pathological effects of calciotropic hormones and proresorptive cytokines.     

Cell intrinsic alterations underlie hematopoietic stem cell aging

Loss of immune function and an increased incidence of myeloid leukemia are two of the most clinically significant consequences of aging of the hematopoietic system. To better understand the mechanisms underlying hematopoietic aging, we evaluated the cell intrinsic functional and molecular properties of highly purified long-term hematopoietic stem cells (LT-HSCs) from young and old mice. We found that LT-HSC aging was accompanied by cell autonomous changes, including increased stem cell self-renewal, differential capacity to generate committed myeloid and lymphoid progenitors, and diminished lymphoid potential. Expression profiling revealed that LT-HSC aging was accompanied by the systemic down-regulation of genes mediating lymphoid specification and function and up-regulation of genes involved in specifying myeloid fate and function. Moreover, LT-HSCs from old mice expressed elevated levels of many genes involved in leukemic transformation. These data support a model in which age-dependent alterations in gene expression at the stem cell level presage downstream developmental potential and thereby contribute to age-dependent immune decline, and perhaps also to the increased incidence of leukemia in the elderly.     

Recombinant human prolactin promotes human beta cell survival via inhibition of extrinsic and intrinsic apoptosis pathways

Aims/hypothesis

Transplantation of pancreatic islets constitutes a promising alternative treatment for type 1 diabetes. However, it is limited by the shortage of organ donors. Previous results from our laboratory have demonstrated beneficial effects of recombinant human prolactin (rhPRL) treatment on beta cell cultures. We therefore investigated the role of rhPRL action in human beta cell survival, focusing on the molecular mechanisms involved in this process.

Methods

Human pancreatic islets were isolated using an automated method. Islet cultures were pre-treated in the absence or presence of rhPRL and then subjected to serum starvation or cytokine treatment. Beta cells were labelled with Newport green and apoptosis was evaluated using flow cytometry analysis. Levels of BCL2 gene family members were studied by quantitative RT-PCR and western blot. Caspase-8, -9 and -3 activity, as well as nitric oxide production, were evaluated by fluorimetric assays.

Results

The proportion of apoptotic beta cells was significantly lowered in the presence of rhPRL under both cell death-induced conditions. We also demonstrated that cytoprotection may involve an increase of BCL2/BAX ratio, as well as inhibition of caspase-8, -9 and -3.

Conclusions/interpretation

Our study provides relevant evidence for a protective effect of lactogens on human beta cell apoptosis. The results also suggest that the improvement of cell survival may involve, at least in part, inhibition of cell death pathways controlled by the BCL2 gene family members. These findings are highly relevant for improvement of the islet isolation procedure and for clinical islet transplantation.

     

Calcium-dependent regulation of the voltage-gated sodium channel hH1: intrinsic and extrinsic sensors use a common molecular switch

The function of the human cardiac voltage-gated sodium channel Na(V)1.5 (hH1) is regulated in part by binding of calcium to an EF hand in the C-terminal cytoplasmic domain. hH1 is also regulated via an extrinsic calcium-sensing pathway mediated by calmodulin (CaM) via binding to an IQ motif immediately adjacent to the EF-hand domain. The intrinsic EF-hand domain is shown here to interact with the IQ motif, which controls calcium affinity. Remarkably, mutation of the IQ residues has only a minor effect on CaM affinity but drastically reduces calcium affinity of the EF-hand domain, whereas the Brugada mutation A1924T significantly reduces CaM affinity but has no effect on calcium affinity of the EF-hand domain. Moreover, the differences in the biochemical effects of the mutations directly correlate with contrasting effects on channel electrophysiology. A comprehensive model is proposed in which the hH1 IQ motif serves as a molecular switch, coupling the intrinsic and extrinsic calcium sensors.     

Placental lactogen-I gene activation in differentiating trophoblast cells: extrinsic and intrinsic regulation involving mitogen-activated protein kinase signaling pathways

Trophoblast giant cells are one of the primary endocrine cell types of the rodent placenta. Placental lactogen-I (PL-I) is the initial prolactin (PRL) family member expressed as trophoblast giant cells differentiate. In this report, we use the Rcho-1 trophoblast cell line as a model for studying the regulation of PL-I gene expression during trophoblast giant cell differentiation. Evidence is provided for trophoblast cell expression of epidermal growth factor receptor (EGFR), ErbB2, fibroblast growth factor receptor 1 (FGFR1), transforming growth factor-alpha, and heparin-binding EGF. EGF and FGF-2 stimulated PL-I mRNA and protein accumulation and PL-I promoter activity in a concentration-dependent manner. These latter growth factor actions on PL-I promoter activities were specifically inhibited by cotransfection with dominant negative constructs for EGFR and FGFRs respectively. Utilization of the mitogen-activated protein kinase (MAPK) pathway by EGF and FGF-2 in trophoblast cells was demonstrated by growth factor stimulation of a Gal4 DNA binding/Elk1 transactivational domain fusion construct, and more specifically by activation of extracellular signal regulated kinase and p38 MAPK. PL-I gene activation was also sensitive to disruption of MAPK and activation protein-1 (AP-1) signaling pathways. In conclusion, autocrine/paracrine pathways involving EGFR and FGFR1, MAPK and AP-1 are shown to participate in the regulation of the PL-I gene in differentiating trophoblast cells.     

细胞炎症反应与脂质代谢的相互作用及调节

本文讨论了脂质代谢紊乱与炎症反应在动脉粥样硬化发生发展中的作用及相互关系。脂质代谢失衡诱发炎症反应;脂蛋白异常修饰和异常定位调节炎症反应,脂质代谢紊乱通过影响单核细胞、M1/M2漂移、核苷酸结合寡聚化结构域样受体含pyrin结构域蛋白3炎症小体等诱导炎症反应。而炎症促进细胞对脂质的摄取和蓄积,抑制细胞脂质流出,与脂代谢紊乱共同促进了动脉粥样硬化的发展。Caveolae/Caveolin、核因子κB通路和过氧化体增殖物激活型受体通路等调节了炎症与脂代谢之间的相互作用。     

Separating intrinsic from extrinsic fluctuations in dynamic biological systems

From molecules in cells to organisms in ecosystems, biological populations fluctuate due to the intrinsic randomness of individual events and the extrinsic influence of changing environments. The combined effect is often too complex for effective analysis, and many studies therefore make simplifying assumptions, for example ignoring either intrinsic or extrinsic effects to reduce the number of model assumptions. Here we mathematically demonstrate how two identical and independent reporters embedded in a shared fluctuating environment can be used to identify intrinsic and extrinsic noise terms, but also how these contributions are qualitatively and quantitatively different from what has been previously reported. Furthermore, we show for which classes of biological systems the noise contributions identified by dual-reporter methods correspond to the noise contributions predicted by correct stochastic models of either intrinsic or extrinsic mechanisms. We find that for broad classes of systems, the extrinsic noise from the dual-reporter method can be rigorously analyzed using models that ignore intrinsic stochasticity. In contrast, the intrinsic noise can be rigorously analyzed using models that ignore extrinsic stochasticity only under very special conditions that rarely hold in biology. Testing whether the conditions are met is rarely possible and the dual-reporter method may thus produce flawed conclusions about the properties of the system, particularly about the intrinsic noise. Our results contribute toward establishing a rigorous framework to analyze dynamically fluctuating biological systems.     

Coordination of intrinsic, extrinsic, and endoplasmic reticulum-mediated apoptosis by imatinib mesylate combined with arsenic trioxide in chronic myeloid leukemia

A treatment strategy that combines arsenic trioxide (ATO) with the tyrosine kinase inhibitor imatinib mesylate (STI571, Gleevec) appears to induce markedly more cell apoptosis than imatinib mesylate alone in chronic myeloid leukemia (CML). To understand the mechanisms underlying the synergistic/additive action of these agents, we applied cDNA microarrays, component plane presentation integrated self-organizing map (CPP-SOM), and methods of protein biochemistry to study cell apoptosis induced by imatinib mesylate, ATO, and the combination of the 2 agents in the CML cell line K562. Numerous features with temporospatial relationships were revealed, indicating the coordinated regulation of molecular networks from various aspects of proapoptotic and apoptotic activities in CML. Imatinib mesylate appears to induce mainly the intrinsic pathway of cell apoptosis, whereas ATO induces the endoplasmic reticulum (ER) stress-mediated pathway of cell apoptosis, and the combination of the 2 agents seems to more effectively induce the intrinsic, extrinsic, and ER stress-mediated pathways of cell apoptosis, which results in a more effective and efficient induction of programmed cell death in K562 cells. This finding appears to be supported also by data derived from bone marrow cells of 2 patients with CML, one in chronic phase and the other in blast-crisis phase of the disease.     

Increase in perforin-positive peripheral blood lymphocytes in extrinsic and intrinsic asthma

The cause of asthma, which has been linked to a chronic, T-cell-mediated bronchial inflammation, remains unclear. A number of other T-lymphocyte-mediated, chronic inflammatory disorders have been associated with autoimmunity and there are data indicating that autoimmune phenomena might also be present in asthma. Expression of perforin, a cytotoxic molecule produced by lymphocytes, has been implicated in the pathogenesis of autoimmune diseases. We therefore tested the hypothesis that allergic and intrinsic asthma might be associated with an increase in lymphocytes producing perforin by comparing the expression of intracellular perforin in peripheral blood lymphocytes of patients with extrinsic asthma (n = 13), intrinsic asthma (n = 7), and healthy control subjects (n = 18). Lymphocytes were identified using flow cytometry and subdivided into CD3(+), CD4(+), CD8(+), CD16(+), and CD56(+) subpopulations after staining with appropriate monoclonal antibodies. The percentage of perforin-positive total lymphocytes was significantly elevated in patients with allergic as well as intrinsic asthma when compared with normal control subjects. Analysis of lymphocyte subpopulations also revealed a significant increase in the percentage of CD3(+), CD4(+), CD8(+), and CD56(+) cells expressing perforin in allergic asthma and a significant increase in the percentage of CD4(+) and CD56(+) cells in intrinsic asthma when compared with healthy control subjects. Perforin expression in CD4(+) cells in intrinsic asthma was also significantly elevated compared with allergic asthma. We conclude that allergic and intrinsic asthma is associated with increased expression of perforin in T-lymphocyte subsets.     

Increase in perforin-positive peripheral blood lymphocytes in extrinsic and intrinsic asthma

The cause of asthma which has been linked to a chronic, T-cell-mediated bronchial inflammation, remains unclear. A number of other T-lymphocyte-mediated, chronic inflammatory disorders have been associated with autoimmunity and there are data indicating that autoimmune phenomena might also be present in asthma. Expression of perforin, a cytotoxic molecule produced by lymphocytes, has been implicated in the pathogenesis of autoimmune disease. We therefore tested the hypothesis that allergic and intrinsic asthma might be associated with an increase in lymphocytes producing perforin by comparing the expression of intracellular perforin in peripheral blood lymphocytes of patients with extrinsic asthma (n = 13), intrinsic asthma (n = 7), and healthy (control subjects (n = 18). Lymphocytes were identified using flow cytometry and subdivided into CD3(+), CD4(+), CD8(+), CD16(+), and CD56(+) subpopulations after staining with appropriate monoclonal antibodies. The percentage of perforin-positive total lymphocytes as significantly elevated in patients with allergic as well as intrinsic asthma when compared with normal control subjects. Analysis of lymphocyte subpopulations also revealed a significant increase in the percentage of CD3(+), CD4(+), CD8(+), and CD56(+) cells expressing perforin in allergic asthma and a significant increase in the percentage of CD4(+), and CD56(+) cells in intrinsic asthma when compare with healthy control subjects. Perforin expression in CD4(+) cells in intrinsic asthma was also significantly elevated compared with allergic asthma. We conclude that allergic and intrinsic asthma is associated with increased expression of perforin in T-lymphocyte subsets.