Role of Cdk4 in lymphocyte function and allergen response

We recently described a new adhesion pathway in lymphocytes that is dependent on Cyclin-dependent kinase (Cdk) 4 activity and mediates lymphocyte interactions with endothelial matrix. We showed that Cdk4^−/− mice had impaired recruitment of lymphocytes following bleomycin model of acute lung injury. In this study, we characterized the development and function of hematopoietic cells in Cdk4^−/− mice and assessed the response of Cdk4^−/− mice to allergen challenge. Cdk4^−/− mice had hypoplastic thymuses with decreased total thymocyte cell numbers and increased CD4/CD8 double negative cells. Cdk4^−/− bone marrow (BM) chimeric mice showed similar findings. Thymocytes from either Cdk4^−/− or Cdk4^−/− BM chimeric mice proliferated equally well as wild type controls in response to IL-2 activation. However Cdk4^−/− thymocytes had decreased adhesion to both endothelial cell matrix and fibronectin compared to wild-type (WT) controls, whereas Cdk4^−/− and WT splenocytes had similar adhesion. When Cdk4^−/− BM chimeric mice and wild type BM chimeric mice were sensitized and challenged by intranasal administration of ovalbumin, we found no differences in allergic responses in the lung and airways between the two groups, as measured by inflammatory cell infiltrate, airway hyperreactivity, IgE levels and cytokine levels. In summary, we show that Cdk4 plays a previously unrecognized role in thymocyte maturation and adhesion, but is not required for thymocyte proliferation. In addition, Cdk4 is not required for lymphocyte trafficking to the lung following allergen sensitization and challenge.Key words: cell adhesion, leukocyte trafficking, thymocyte development     

Genetic Characterization of the Role of the Cip/Kip Family of Proteins as Cyclin-Dependent Kinase Inhibitors and Assembly Factors

The Cip/Kip family, namely, p21^Cip1, p27^Kip1, and p57^Kip2, are stoichiometric cyclin-dependent kinase inhibitors (CKIs). Paradoxically, they have been proposed to also act as positive regulators of Cdk4/6-cyclin D by stabilizing these heterodimers. Loss of p21^Cip1 and p27^Kip1 reduces Cdk4/6-cyclin D complexes, although with limited phenotypic consequences compared to the embryonic lethality of Cdk4/6 or triple cyclin D deficiency. This milder phenotype was attributed to Cdk2 compensatory mechanisms. To address this controversy using a genetic approach, we generated Cdk2^−/− p21^−/− p27^−/− mice. Triple-knockout mouse embryonic fibroblasts (MEFs) displayed minimal levels of D-type cyclins and Cdk4/6-cyclin D complexes. p57^Kip2 downregulation in the absence of p21^Cip1 and p27^Kip1 aggravated this phenotype, yet MEFs lacking all Cip/Kip proteins exhibited increased retinoblastoma phosphorylation, together with enhanced proliferation and transformation capacity. In vivo, Cdk2 ablation induced partial perinatal lethality in p21^−/− p27^−/− mice, suggesting partial Cdk2-dependent compensation. However, Cdk2^−/− p21^−/− p27^−/− survivors displayed all phenotypes described for p27^−/− mice, including organomegalia and pituitary tumors. Thus, Cip/Kip deficiency does not impair interphasic Cdk activity even in the absence of Cdk2, suggesting that their Cdk-cyclin assembly function is dispensable for homeostatic control in most cell types.     

Reduced EGFR signaling in progenitor cells of the adult subventricular zone attenuates oligodendrogenesis after demyelination

Neural progenitor cells that express the NG2 proteoglycan are present in different regions of the adult mammalian brain where they display distinct morphologies and proliferative rates. In the developing postnatal and adult mouse, NG2(+) cells represent a major cell population of the subventricular zone (SVZ). NG2(+) cells divide in the anterior and lateral region of the SVZ, and are stimulated to proliferate and migrate out of the SVZ by focal demyelination of the corpus callosum (CC). Many NG2(+) cells are labeled by GFP-retrovirus injection into the adult SVZ, demonstrating that NG2(+) cells actively proliferate under physiological conditions and after demyelination. Under normal physiological conditions and after focal demyelination, proliferation of NG2(+) cells is significantly attenuated in wa2 mice, which are characterized by reduced signaling of the epidermal growth factor receptor (EGFR). This results in reduced SVZ-to-lesion migration of NG2(+) cells and oligodendrogenesis in the lesion. Expression of vascular endothelial growth factor (VEGF) and EGFR ligands, such as heparin binding-EGF and transforming growth factor alpha, is upregulated in the SVZ after focal demyelination of the CC. EGF-induced oligodendrogenesis and myelin protein expression in wild-type SVZ cells in culture are significantly attenuated in wa2 SVZ cells. Our results demonstrate that the response of NG2(+) cells in the SVZ and their subsequent differentiation in CC after focal demyelination depend on EGFR signaling.     

Adult Mouse Subventricular Zone Stem and Progenitor Cells Are Sessile and Epidermal Growth Factor Receptor Negatively Regulates Neuroblast Migration

Background

The adult subventricular zone (SVZ) contains stem and progenitor cells that generate neuroblasts throughout life. Although it is well accepted that SVZ neuroblasts are migratory, recent evidence suggests their progenitor cells may also exhibit motility. Since stem and progenitor cells are proliferative and multipotential, if they were also able to move would have important implications for SVZ neurogenesis and its potential for repair.

Methodology/Principal Findings

We studied whether SVZ stem and/or progenitor cells are motile in transgenic GFP+ slices with two photon time lapse microscopy and post hoc immunohistochemistry. We found that stem and progenitor cells; mGFAP-GFP+ cells, bright nestin-GFP+ cells and Mash1+ cells were stationary in the SVZ and rostral migratory stream (RMS). In our search for motile progenitor cells, we uncovered a population of motile βIII-tubulin+ neuroblasts that expressed low levels of epidermal growth factor receptor (EGFr). This was intriguing since EGFr drives proliferation in the SVZ and affects migration in other systems. Thus we examined the potential role of EGFr in modulating SVZ migration. Interestingly, EGFr^low neuroblasts moved slower and in more tortuous patterns than EGFr-negative neuroblasts. We next questioned whether EGFr stimulation affects SVZ cell migration by imaging Gad65-GFP+ neuroblasts in the presence of transforming growth factor alpha (TGF-α), an EGFr-selective agonist. Indeed, acute exposure to TGF-α decreased the percentage of motile cells by approximately 40%.

Conclusions/Significance

In summary, the present study directly shows that SVZ stem and progenitor cells are static, that EGFr is retained on some neuroblasts, and that EGFr stimulation negatively regulates migration. This result suggests an additional role for EGFr signaling in the SVZ.     

Targeted Ablation of miR-21 Decreases Murine Eosinophil Progenitor Cell Growth

MiR-21 is one of the most up-regulated miRNAs in multiple allergic diseases associated with eosinophilia and has been shown to positively correlate with eosinophil levels. Herein, we show that miR-21 is up-regulated during IL-5-driven eosinophil differentiation from progenitor cells in vitro. Targeted ablation of miR-21 leads to reduced eosinophil progenitor cell growth. Furthermore, miR-21^−/− eosinophil progenitor cells have increased apoptosis as indicated by increased levels of annexin V positivity compared to miR-21^+/+ eosinophil progenitor cells. Indeed, miR-21^−/− mice have reduced blood eosinophil levels in vivo and reduced eosinophil colony forming unit capacity in the bone marrow. Using gene expression microarray analysis, we identified dysregulation of genes involved in cell proliferation (e,g, Ms4a3, Grb7), cell cycle and immune response as the most significant pathways affected by miR-21 in eosinophil progenitors. These results demonstrate that miR-21 can regulate the development of eosinophils by influencing eosinophil progenitor cell growth. Our findings have identified one of the first miRNAs with a role in regulating eosinophil development.     

Generation and Characterization of Induced Pluripotent Stem Cells from Aid-Deficient Mice

It has been shown that DNA demethylation plays a pivotal role in the generation of induced pluripotent stem (iPS) cells. However, the underlying mechanism of this action is still unclear. Previous reports indicated that activation-induced cytidine deaminase (Aid, also known as Aicda) is involved in DNA demethylation in several developmental processes, as well as cell fusion-mediated reprogramming. Based on these reports, we hypothesized that Aid may be involved in the DNA demethylation that occurs during the generation of iPS cells. In this study, we examined the function of Aid in iPS cell generation using Aid knockout (Aid^−/−) mice expressing a GFP reporter under the control of a pluripotent stem cell marker, Nanog. By introducing Oct3/4, Sox2, Klf4 and c-Myc, Nanog-GFP-positive iPS cells could be generated from the fibroblasts and primary B cells of Aid^−/− mice. Their induction efficiency was similar to that of wild-type (Aid^+/+) iPS cells. The Aid^−/− iPS cells showed normal proliferation and gave rise to chimeras, indicating their capacity for self-renewal and pluripotency. A comprehensive DNA methylation analysis showed only a few differences between Aid^+/+ and Aid^−/− iPS cells. These data suggest that Aid does not have crucial functions in DNA demethylation during iPS cell generation.     

p19Ink4d Is a Tumor Suppressor and Controls Pituitary Anterior Lobe Cell Proliferation

Pituitary tumors develop in about one-quarter of the population, and most arise from the anterior lobe (AL). The pituitary gland is particularly sensitive to genetic alteration of genes involved in the cyclin-dependent kinase (CDK) inhibitor (CKI)–CDK-retinoblastoma protein (Rb) pathway. Mice heterozygous for the Rb mutation develop pituitary tumors, with about 20% arising from the AL. Perplexingly, none of the CKI-deficient mice reported thus far develop pituitary AL tumors. In this study, we show that deletion of p19^Ink4d (p19), a CKI gene, in mice results in spontaneous development of tumors in multiple organs and tissues. Specifically, more than one-half of the mutant mice developed pituitary hyperplasia or tumors predominantly in the AL. Tumor development is associated with increased cell proliferation and enhanced activity of Cdk4 and Cdk6 and phosphorylation of Rb protein. Though Cdk4 is indispensable for postnatal pituitary cell proliferation, it is not required for the hyperproliferative pituitary phenotype caused by p19 loss. Loss of p19 phosphorylates Rb in Cdk4^−/− pituitary AL cells and mouse embryonic fibroblasts (MEFs) and rescues their proliferation defects, at least partially, through the activation of Cdk6. These results provide the first genetic evidence that p19 is a tumor suppressor and the major CKI gene that controls pituitary AL cell proliferation.     

Akhirin regulates the proliferation and differentiation of neural stem cells/progenitor cells at neurogenic niches in mouse brain

Specialized microenvironment, or neurogenic niche, in embryonic and postnatal mouse brain plays critical roles during neurogenesis throughout adulthood. The subventricular zone (SVZ) and the dentate gyrus (DG) of hippocampus in the mouse brain are two major neurogenic niches where neurogenesis is directed by numerous regulatory factors. Now, we report Akhirin (AKH), a stem cell maintenance factor in mouse spinal cord, plays a pivotal regulatory role in the SVZ and in the DG. AKH showed specific distribution during development in embryonic and postnatal neurogenic niches. Loss of AKH led to abnormal development of the ventricular zone and the DG along with reduction of cellular proliferation in both regions. In AKH knockout mice (AKH^−/−), quiescent neural stem cells (NSCs) increased, while proliferative NSCs or neural progenitor cells decreased at both neurogenic niches. In vitro NSC culture assay showed increased number of neurospheres and reduced neurogenesis in AKH^−/−. These results indicate that AKH, at the neurogenic niche, exerts dynamic regulatory role on NSC self-renewal, proliferation and differentiation during SVZ and hippocampal neurogenesis.     

Tripartite Motif-Containing Protein 30 Modulates TCR-Activated Proliferation and Effector Functions in CD4+ T Cells

To avoid excessive activation, immune signals are tightly controlled by diverse inhibitory proteins. TRIM30, a tripartite motif (TRIM)-containing protein is one of such inhibitors known to function in macrophages. To define the roles of TRIM30, we generated Trim30 knockout (Trim30 ^−/−) mice. Trim30 deletion caused no major developmental defects in any organs, nor showed any discernable defect in the activation of macrophages. But, Trim30 ^−/− mice showed increased CD4/CD8 ratio when aged and Trim30 ^−/− CD4⁺ T cells exhibited an abnormal response upon TCR activation, in particular in the absence of a costimulatory signal. Adoptive transfer of wild-type and Trim30 ^−/− CD4⁺ T cells together into lymphopenic hosts confirmed higher proliferation of the Trim30 ^−/− CD4⁺ T cells in vivo. Despite the enhanced proliferation, Trim30 ^−/− T cells showed decreased levels of NF-κB activation and IL-2 production compared to wild-type cells. These results indicate a distinct requirement for TRIM30 in modulation of NF-κB activation and cell proliferation induced by TCR stimulation.     

Comparison of Cortical and White Matter Traumatic Brain Injury Models Reveals Differential Effects in the Subventricular Zone and Divergent Sonic Hedgehog Signaling Pathways in Neuroblasts and Oligodendrocyte Progenitors

The regenerative capacity of the central nervous system must be optimized to promote repair following traumatic brain injury (TBI) and may differ with the site and form of damage. Sonic hedgehog (Shh) maintains neural stem cells and promotes oligodendrogenesis. We examined whether Shh signaling contributes to neuroblast (doublecortin) or oligodendrocyte progenitor (neural/glial antigen 2 [NG2]) responses in two distinct TBI models. Shh-responsive cells were heritably labeled in vivo using Gli1-CreER^T2;R26-YFP bitransgenic mice with tamoxifen administration on Days 2 and 3 post-TBI. Injury to the cerebral cortex was produced with mild controlled cortical impact. Yellow fluorescent protein (YFP) cells decreased in cortical lesions. Total YFP cells increased in the subventricular zone (SVZ), indicating Shh pathway activation in SVZ cells, including doublecortin-labeled neuroblasts. The alternate TBI model produced traumatic axonal injury in the corpus callosum. YFP cells decreased within the SVZ and were rarely double labeled as NG2 progenitors. NG2 progenitors increased in the cortex, with a similar pattern in the corpus callosum. To further test the potential of NG2 progenitors to respond through Shh signaling, Smoothened agonist was microinjected into the corpus callosum to activate Shh signaling. YFP cells and NG2 progenitors increased in the SVZ but were not double labeled. This result indicates that either direct Smoothened activation in NG2 progenitors does not signal through Gli1 or that Smoothened agonist acts indirectly to increase NG2 progenitors. Therefore, in all conditions, neuroblasts exhibited differential Shh pathway utilization compared with oligodendrocyte progenitors. Notably, cortical versus white matter damage from TBI produced opposite responses of Shh-activated cells within the SVZ.     

Cyclin B1/Cdk1 Phosphorylation of Mitochondrial p53 Induces Anti-Apoptotic Response

The pro-apoptotic function of p53 has been well defined in preventing genomic instability and cell transformation. However, the intriguing fact that p53 contributes to a pro-survival advantage of tumor cells under DNA damage conditions raises a critical question in radiation therapy for the 50% human cancers with intact p53 function. Herein, we reveal an anti-apoptotic role of mitochondrial p53 regulated by the cell cycle complex cyclin B1/Cdk1 in irradiated human colon cancer HCT116 cells with p53^+/+ status. Steady-state levels of p53 and cyclin B1/Cdk1 were identified in the mitochondria of many human and mouse cells, and their mitochondrial influx was significantly enhanced by radiation. The mitochondrial kinase activity of cyclin B1/Cdk1 was found to specifically phosphorylate p53 at Ser-315 residue, leading to enhanced mitochondrial ATP production and reduced mitochondrial apoptosis. The improved mitochondrial function can be blocked by transfection of mutant p53 Ser-315-Ala, or by siRNA knockdown of cyclin B1 and Cdk1 genes. Enforced translocation of cyclin B1 and Cdk1 into mitochondria with a mitochondrial-targeting-peptide increased levels of Ser-315 phosphorylation on mitochondrial p53, improved ATP production and decreased apoptosis by sequestering p53 from binding to Bcl-2 and Bcl-xL. Furthermore, reconstitution of wild-type p53 in p53-deficient HCT116 p53^−/− cells resulted in an increased mitochondrial ATP production and suppression of apoptosis. Such phenomena were absent in the p53-deficient HCT116 p53^−/− cells reconstituted with the mutant p53. These results demonstrate a unique anti-apoptotic function of mitochondrial p53 regulated by cyclin B1/Cdk1-mediated Ser-315 phosphorylation in p53-wild-type tumor cells, which may provide insights for improving the efficacy of anti-cancer therapy, especially for tumors that retain p53.     

p21Waf1/Cip1 Inhibition of Cyclin E/Cdk2 Activity Prevents Endoreduplication after Mitotic Spindle Disruption

During a normal cell cycle, entry into S phase is dependent on completion of mitosis and subsequent activation of cyclin-dependent kinases (Cdks) in G₁. These events are monitored by checkpoint pathways. Recent studies and data presented herein show that after treatment with microtubule inhibitors (MTIs), cells deficient in the Cdk inhibitor p21^Waf1/Cip1 enter S phase with a ≥4N DNA content, a process known as endoreduplication, which results in polyploidy. To determine how p21 prevents MTI-induced endoreduplication, the G₁/S and G₂/M checkpoint pathways were examined in two isogenic cell systems: HCT116 p21^+/+ and p21^−/− cells and H1299 cells containing an inducible p21 expression vector (HIp21). Both HCT116 p21^−/− cells and noninduced HIp21 cells endoreduplicated after MTI treatment. Analysis of G₁-phase Cdk activities demonstrated that the induction of p21 inhibited endoreduplication through direct cyclin E/Cdk2 regulation. The kinetics of p21 inhibition of cyclin E/Cdk2 activity and binding to proliferating-cell nuclear antigen in HCT116 p21^+/+ cells paralleled the onset of endoreduplication in HCT116 p21^−/− cells. In contrast, loss of p21 did not lead to deregulated cyclin D1-dependent kinase activities, nor did p21 directly regulate cyclin B1/Cdc2 activity. Furthermore, we show that MTI-induced endoreduplication in p53-deficient HIp21 cells was due to levels of p21 protein below a threshold required for negative regulation of cyclin E/Cdk2, since ectopic expression of p21 restored cyclin E/Cdk2 regulation and prevented endoreduplication. Based on these findings, we propose that p21 plays an integral role in the checkpoint pathways that restrain normal cells from entering S phase after aberrant mitotic exit due to defects in microtubule dynamics.     

Robo4 Plays a Role in Bone Marrow Homing and Mobilization,but Is Not Essential in the Long-Term Repopulating Capacity of Hematopoietic Stem Cells

Roundabout (Robo) family proteins are immunoglobulin-type surface receptors critical for cellular migration and pathway finding of neuronal axons. We have previously shown that Robo4 was specifically expressed in hematopoietic stem and progenitor cells and its high expression correlated with long-term repopulating (LTR) capacity. To reveal the physiological role of Robo4 in hematopoiesis, we examined the effects of Robo4 disruption on the function of hematopoietic stem cells (HSCs) and progenitors. In Robo4-deficient mice, basic hematological parameters including complete blood cell count and differentiation profile were not affected. In contrast to the previous report, HSC/hematopoietic progenitor (HPC) frequencies in the bone marrow (BM) were perfectly normal in Robo4^−/− mice. Moreover, Robo4^−/− HSCs were equally competitive as wild-type HSCs in transplantation assays and had normal long-term repopulating (LTR) capacity. Of note, the initial engraftment at 4-weeks after transplantation was slightly impaired by Robo4 ablation, suggesting a marginal defect in BM homing of Robo4^−/− HSCs. In fact, homing efficiencies of HSCs/HPCs to the BM was significantly impaired in Robo4-deficient mice. On the other hand, granulocyte-colony stimulating factor-induced peripheral mobilization of HSCs was also impaired by Robo4 disruption. Lastly, marrow recovery from myelosuppressive stress was equally efficient in WT- and Robo4-mutant mice. These results clearly indicate that Robo4 plays a role in HSC trafficking such as BM homing and peripheral mobilization, but is not essential in the LTR and self-renewal capacity of HSCs.     

Erythropoietin Amplifies Stroke-Induced Oligodendrogenesis in the Rat

Background

Erythropoietin (EPO), a hematopoietic cytokine, enhances neurogenesis and angiogenesis during stroke recovery. In the present study, we examined the effect of EPO on oligodendrogenesis in a rat model of embolic focal cerebral ischemia.

Methodology and Principal Findings

Recombinant human EPO (rhEPO) at a dose of 5,000 U/kg (n = 18) or saline (n = 18) was intraperitoneally administered daily for 7 days starting 24 h after stroke onset. Treatment with rhEPO augmented actively proliferating oligodendrocyte progenitor cells (OPCs) measured by NG2 immunoreactive cells within the peri-infarct white matter and the subventricular zone (SVZ), but did not protect against loss of myelinating oligodendrocytes measured by cyclic nucleotide phosphodiesterase (CNPase) positive cells 7 days after stroke. However, 28 and 42 days after stroke, treatment with rhEPO significantly increased myelinating oligodendrocytes and myelinated axons within the peri-infarct white matter. Using lentivirus to label subventricular zone (SVZ) neural progenitor cells, we found that in addition to the OPCs generated in the peri-infarct white matter, SVZ neural progenitor cells contributed to rhEPO-increased OPCs in the peri-infarct area. Using bromodeoxyuridine (BrdU) for birth-dating cells, we demonstrated that myelinating oligodendrocytes observed 28 days after stroke were derived from OPCs. Furthermore, rhEPO significantly improved neurological outcome 6 weeks after stroke. In vitro, rhEPO increased differentiation of adult SVZ neural progenitor cells into oligodendrocytes and enhanced immature oligodendrocyte cell proliferation.

Conclusions

Our in vivo and in vitro data indicate that EPO amplifies stroke-induced oligodendrogenesis that could facilitate axonal re-myelination and lead to functional recovery after stroke.     

Adult mesenchymal stem cell ageing interplays with depressed mitochondrial Ndufs6

Mesenchymal stem cell (MSC)-based therapy has emerged as a novel strategy to treat many degenerative diseases. Accumulating evidence shows that the function of MSCs declines with age, thus limiting their regenerative capacity. Nonetheless, the underlying mechanisms that control MSC ageing are not well understood. We show that compared with bone marrow-MSCs (BM-MSCs) isolated from young and aged samples, NADH dehydrogenase (ubiquinone) iron-sulfur protein 6 (Ndufs6) is depressed in aged MSCs. Similar to that of Ndufs6 knockout (Ndufs6^−/−) mice, MSCs exhibited a reduced self-renewal and differentiation capacity with a tendency to senescence in the presence of an increased p53/p21 level. Downregulation of Ndufs6 by siRNA also accelerated progression of wild-type BM-MSCs to an aged state. In contrast, replenishment of Ndufs6 in Ndufs6^−/−-BM-MSCs significantly rejuvenated senescent cells and restored their proliferative ability. Compared with BM-MSCs, Ndufs6^−/−-BM-MSCs displayed increased intracellular and mitochondrial reactive oxygen species (ROS), and decreased mitochondrial membrane potential. Treatment of Ndufs6^−/−-BM-MSCs with mitochondrial ROS inhibitor Mito-TEMPO notably reversed the cellular senescence and reduced the increased p53/p21 level. We provide direct evidence that impairment of mitochondrial Ndufs6 is a putative accelerator of adult stem cell ageing that is associated with excessive ROS accumulation and upregulation of p53/p21. It also indicates that manipulation of mitochondrial function is critical and can effectively protect adult stem cells against senescence.Subject terms: Ageing, Stem-cell research