Miguel R. Campanero

Control of Cyclin G2 mRNA Expression by Forkhead Transcription Factors: Novel Mechanism for Cell Cycle Control by Phosphoinositide 3-Kinase and Forkhead

ABSTRACT Cyclin G2 is an unconventional cyclin highly expressed in postmitotic cells. Unlike classical cyclins that promote cell cycle progression, cyclin G2 blocks cell cycle entry. Here we studied the mechanisms that regulate cyclin G2 mRNA expression during the cell cycle. Analysis of synchronized NIH 3T3 cell cultures showed elevated cyclin G2 mRNA expression levels at G0, with a considerable reduction as cells enter cell cycle. Downregulation of cyclin G2 mRNA levels requires activation of phosphoinositide 3-kinase, suggesting that this enzyme controls cyclin G2 mRNA expression. Because the phosphoinositide 3-kinase pathway inhibits the FoxO family of forkhead transcription factors, we examined the involvement of these factors in the regulation of cyclin G2 expression. We show that active forms of the forkhead transcription factor FoxO3a (FKHRL1) increase cyclin G2 mRNA levels. Cyclin G2 has forkhead consensus motifs in its promoter, which are transactivated by constitutive active FoxO3a forms. Finally, interference with forkhead-mediated transcription by overexpression of an inactive form decreases cyclin G2 mRNA expression levels. These results show that FoxO genes regulate cyclin G2 expression, illustrating a new role for phosphoinositide 3-kinase and FoxO transcription factors in the control of cell cycle entry.

Prevention of in vitro neutrophil-endothelial attachment through shedding of L-selectin by nonsteroidal antiinflammatory drugs.

The activation of the endothelial cells by extravascular stimuli is the key event in the extravasation of circulating leukocytes to target tissues. L-selectin, a member of the selectin family, is constitutively expressed by white cells, and is the molecule involved in the initial binding of leukocytes to activated endothelium. After activation, leukocytes rapidly release L-selectin from the cell surface, suggesting that the functional activity of this molecule is controlled in large part by its appearance and disappearance from cell surface. We have studied in a neutrophil-activated endothelial cell binding assay, the effect of different antiinflammatory drugs (steroidal and nonsteroidal) in the L-selectin-mediated interaction of neutrophils with activated endothelial cells. Some nonsteroidal antiinflammatory drugs (NSAIDs), such as indomethacin, diclofenac, ketoprofen, and aspirin, but not steroids, strongly inhibited the neutrophil-endothelial cell attachment. Furthermore, we also investigated the underlying mechanism of this functional effect. The expression of L-selectin on the neutrophil surface rapidly decreased in the presence of different NSAIDs, in a dose- and time-dependent manner, whereas no changes in the expression of other adhesion molecules such as CD11a, CD11b, CD31, or ICAM-3 (CD50) were observed. Interestingly, studies in vivo on healthy volunteers treated with physiological doses of indomethacin showed a significant decrease of L-selectin neutrophil expression. Only diclofenac induced an upregulation of CD11b expression, suggesting an activating effect on neutrophils. No enzyme release was observed upon treatment of neutrophils with different NSAIDs, indicating a lack of degranulatory activity of NSAIDs, with the exception of diclofenac. The downregulation of L-selectin expression was due to the rapid cleavage and shedding of the membrane L-selectin, as determined by both immunoprecipitation from 125I-labeled neutrophils, and quantitative estimation in cell-free supernatants. These results suggest that NSAIDs exert a specific action on adhesion receptor expression in neutrophils, which might account, at least in part, for the antiinflammatory activities of NSAIDs.

E2F1 expression is deregulated and plays an oncogenic role in sporadic Burkitt’s lymphoma

Current treatments of sporadic Burkitts lymphoma (sBL) are associated with severe toxicities. A better understanding of sBL formation would facilitate development of less toxic therapies. The etiology of sBL remains, however, largely unknown, C-MYC up-regulation being the only lesion known to occur in all sBL cases. Several studies examining the role of C-MYC in the pathogenesis of BL have concluded that C-MYC translocation is not the only critical event and that additional unidentified factors are expected to be involved in the formation of this tumor. We herein report that a gene distinct from C-MYC, E2F1, is involved in the formation of all or most sBL tumors. We found that E2F1 is highly expressed in Burkitts lymphoma cell lines and sBL lymphoma specimens. Our data indicate that its elevated expression is not merely the consequence of the presence of more cycling cells in this tumor relative to other cell lines or to other neoplasias. In fact, we show that reduction of its expression in sBL cells inhibits tumor formation and decreases their proliferation rate. We also provide data suggesting that E2F1 collaborates with C-MYC in sBL formation. E2F1 expression down-regulation did not affect, however, the proliferation of human primary diploid fibroblasts. Because E2F1 is not needed for cell proliferation of normal cells, our results reveal E2F1 as a promising therapeutic target for sBL.

Nitric oxide mediates aortic disease in mice deficient in the metalloprotease Adamts1 and in a mouse model of Marfan syndrome

Heritable thoracic aortic aneurysms and dissections (TAAD), including Marfan syndrome (MFS), currently lack a cure, and causative mutations have been identified for only a fraction of affected families. Here we identify the metalloproteinase ADAMTS1 and inducible nitric oxide synthase (NOS2) as therapeutic targets in individuals with TAAD. We show that Adamts1 is a major mediator of vascular homeostasis, given that genetic haploinsufficiency of Adamts1 in mice causes TAAD similar to MFS. Aortic nitric oxide and Nos2 levels were higher in Adamts1-deficient mice and in a mouse model of MFS (hereafter referred to as MFS mice), and Nos2 inactivation protected both types of mice from aortic pathology. Pharmacological inhibition of Nos2 rapidly reversed aortic dilation and medial degeneration in young Adamts1-deficient mice and in young or old MFS mice. Patients with MFS showed elevated NOS2 and decreased ADAMTS1 protein levels in the aorta. These findings uncover a possible causative role for the ADAMTS1–NOS2 axis in human TAAD and warrant evaluation of NOS2 inhibitors for therapy.

A major role for RCAN1 in atherosclerosis progression

Atherosclerosis is a complex inflammatory disease involving extensive vascular vessel remodelling and migration of vascular cells. As RCAN1 is implicated in cell migration, we investigated its contribution to atherosclerosis. We show RCAN1 induction in atherosclerotic human and mouse tissues. Rcan1 was expressed in lesional macrophages, endothelial cells and vascular smooth muscle cells and was induced by treatment of these cells with oxidized LDLs (oxLDLs). Rcan1 regulates CD36 expression and its genetic inactivation reduced atherosclerosis extension and severity in Apoe−/− mice. This effect was mechanistically linked to diminished oxLDL uptake, resistance to oxLDL‐mediated inhibition of macrophage migration and increased lesional IL‐10 and mannose receptor expression. Moreover, Apoe−/−Rcan1−/− macrophages expressed higher‐than‐Apoe−/− levels of anti‐inflammatory markers. We previously showed that Rcan1 mediates aneurysm development and that its expression is not required in haematopoietic cells for this process. However, transplantation of Apoe−/−Rcan1−/− bone‐marrow (BM) cells into Apoe−/− recipients confers atherosclerosis resistance. Our data define a major role for haematopoietic Rcan1 in atherosclerosis and suggest that therapies aimed at inhibiting RCAN1 expression or function might significantly reduce atherosclerosis burden.

Kidins220 accumulates with tau in human Alzheimer's disease and related models: modulation of its calpain-processing by GSK3β/PP1 imbalance

Failures in neurotrophic support and signalling play key roles in Alzheimers disease (AD) pathogenesis. We previously demonstrated that downregulation of the neurotrophin effector Kinase D interacting substrate (Kidins220) by excitotoxicity and cerebral ischaemia contributed to neuronal death. This downregulation, triggered through overactivation of N-methyl-D-aspartate receptors (NMDARs), involved proteolysis of Kidins220 by calpain and transcriptional inhibition. As excitotoxicity is at the basis of AD aetiology, we hypothesized that Kidins220 might also be downregulated in this disease. Unexpectedly, Kidins220 is augmented in necropsies from AD patients where it accumulates with hyperphosphorylated tau. This increase correlates with enhanced Kidins220 resistance to calpain processing but no higher gene transcription. Using AD brain necropsies, glycogen synthase kinase 3-β (GSK3β)-transgenic mice and cell models of AD-related neurodegeneration, we show that GSK3β phosphorylation decreases Kidins220 susceptibility to calpain proteolysis, while protein phosphatase 1 (PP1) action has the opposite effect. As altered activities of GSK3β and phosphatases are involved in tau aggregation and constitute hallmarks in AD, a GSK3β/PP1 imbalance may also contribute to Kidins220 decreased clearance, accumulation and hampered neurotrophin signalling from early stages of the disease pathogenesis. These results encourage searches for mutations in Kidins220 gene and their possible associations to dementias. Finally, our data support a model where the effects of excitotoxicity drastically differ when occurring in cerebral ischaemia versus progressively sustained toxicity along AD progression. The striking differences in Kidins220 stability resulting from chronic versus acute brain damage may also have important implications for the therapeutic intervention of neurodegenerative disorders.

B Lymphocyte Commitment Program Is Driven by the Proto-Oncogene c-myc

c-Myc, a member of the Myc family of transcription factors, is involved in numerous biological functions including the regulation of cell proliferation, differentiation, and apoptosis in various cell types. Of all of its functions, the role of c-Myc in cell differentiation is one of the least understood. We addressed the role of c-Myc in B lymphocyte differentiation. We found that c-Myc is essential from early stages of B lymphocyte differentiation in vivo and regulates this process by providing B cell identity via direct transcriptional regulation of the ebf-1 gene. Our data show that c-Myc influences early B lymphocyte differentiation by promoting activation of B cell identity genes, thus linking this transcription factor to the EBF-1/Pax-5 pathway.

Structure-function analysis of the human integrin VLA-4 (α4/β1): Correlation of proteolytic α4 peptides with α4 epitopes and sites of ligand interaction

The structure-function relationship of the human integrin VLA-4 (alpha 4/beta 1; CD49d/CD29), has been studied in the human B-cell line Ramos by immunochemical and functional analysis. Ramos cells expressed the 150-kDa non-proteolyzed form of the alpha 4 chain, which could be digested upon mild trypsin treatment to generate the 80- and 65-kDa proteolyzed forms, as well as alpha 4 polypeptides of 55 and 50 kDa. In addition, treatment of Ramos cells with high doses of pronase predominantly yielded the 55- and 50-kDa alpha 4 peptides. The trypsin-generated 80- and 65-kDa alpha 4 polypeptides, but not the 55- and 50-kDa fragments, were able to associate with the beta 1 chain. Distinct anti-VLA-4 mAb against four different alpha 4 epitopes, referred to as epitopes A, B1, B2, and C, recognized the 150-kDa alpha 4 chain both associated or non-associated with the beta 1 chain. The alpha 4 proteolytic forms of 80, 65 and 50 kDa were precipitated by the anti-alpha 4 mAb directed against the four different alpha 4 epitopes. On the other hand, the 55-kDa alpha 4 peptide was present in precipitates from anti-alpha 4 mAb specific for epitopes A, B1 and C, but absent in precipitates from the anti-alpha 4 mAb specific for epitope B2. The different adhesive capacities of the VLA-4 integrin, namely the interaction with a 38-kDa fibronectin fragment containing the CS-1 region of plasma fibronectin (Fn-38), the binding to the vascular cell adhesion molecule-1 (VCAM-1), or the ability to mediate the anti-alpha 4-induced cell aggregation, were not altered on VLA-4 from cells upon mild trypsin treatment, when compared to non-treated cells. However, the 55- and 50-kDa alpha 4 forms generated by high-dose pronase cell treatment, failed to mediate cell interaction with Fn-38 or VCAM-1 ligands, and cell aggregation could not be triggered through VLA-4 under these conditions.The structure‐function relationship of the human integrin VLA‐4 (α4/β1; CD49d/CD29), has been studied in the human B‐cell line Ramos by immunochemical and functional analysis. Ramos cells expressed the 150‐kDa non‐proteolyzed form of the α4 chain, which could be digested upon mild trypsin treatment to generate the 80‐ and 65‐kDa proteolyzed forms, as well as α4 polypeptides of 55 and 50 kDa. In addition, treatment of Ramos cells with high doses of pronase predominantly yielded the 55‐ and 50‐kDa α4 peptides. The trypsin‐generated 80‐ and 65‐kDa α4 polypeptides, but not the 55‐ and 50‐kDa fragments, were able to associate with the β1 chain. Distinct anti‐VLA‐4 mAb against four different α4 epitopes, referred to as epitopes A, B1, B2, and C, recognized the 150‐kDa α4 chain both associated or non‐associated with the β1 chain. The α4 proteolytic forms of 80, 65 and 50 kDa were precipitated by the anti‐α4 mAb directed against the four different α4 epitopes. On the other hand, the 55‐kDa α4 peptide was present in precipitates from anti‐α4 mAb specific for epitopes A, B1 and C, but absent in precipitates from the anti‐α4 mAb specific for epitope B2. The different adhesive capacities of the VLA‐4 integrin, namely the interaction with a 38‐kDa fibronectin fragment containing the CS‐1 region of plasma fibronectin (Fn‐38), the binding to the vascular cell adhesion molecule‐1 (VCAM‐1), or the ability to mediate the anti‐α4‐induced cell aggregation, were not altered on VLA‐4 from cells upon mild trypsin treatment, when compared to non‐treated cells. However, the 55‐ and 50‐kDa α4 forms generated by high‐dose pronase cell treatment, failed to mediate cell interaction with Fn‐38 or VCAM‐1 ligands, and cell aggregation could not be triggered through VLA‐4 under these conditions.

The neuronal protein Kidins220/ARMS associates with ICAM-3 and other uropod components and regulates T-cell motility

Kinase D interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat‐rich membrane spanning (ARMS), is a protein that is mainly expressed in brain and neural cells where its function is only starting to be characterized. Here, we show that Kidins220/ARMS is also expressed in T lymphocytes where it is highly concentrated at the uropod of polarized T cells. In this cellular model, Kidins220/ARMS colocalizes with typical uropod T‐cell molecules and coimmunoprecipitates with ICAM‐3. Furthermore, Kidins220/ARMS associates with raft domains at the uropod and coimmunoprecipitates with caveolin‐1, a molecule we show here to be also expressed in T cells. Importantly, induction of morphological polarization in primary T lymphocytes and Jurkat cells enhances Kidins220/ARMS colocalization with ICAM‐3. Conversely, disruption of cell polarity provokes Kidins220/ARMS redistribution from the uropod to other cellular regions and drastically impairs its association with ICAM‐3 in a protein kinase C‐dependent manner. Finally, Kidins220/ARMS knockdown in human polarized T‐cell lines promotes both basal and stromal cell‐derived factor‐1α‐induced directed migration, identifying a novel function for this molecule. Altogether, our findings show that Kidins220/ARMS is a novel component of the uropod involved in the regulation of T‐cell motility, an essential process for the immune response.

Nonlinear Optical 3-Dimensional Method for Quantifying Atherosclerosis Burden

Changes in atherosclerosis burden provide an experimental measure of the effectiveness of therapeutic strategies. Unfortunately, none of the methods used in mice to assess atherosclerosis burden ex vivo is sufficiently accurate and fast enough to process the number of samples required in preclinical studies. We aimed to design an easy-to-implement and relatively fast method for accurate volumetric quantification of atheroma plaque burden in mice. The apolipoprotein E–deficient mouse ( Apoe −/−) is a widely used preclinical model that reproducibly develops hypercholesterolemia and atherosclerosis.1 Two widely used methods for ex vivo assessment of atherosclerosis burden in mice are histopathologic analysis2 and the en face method.3 The histological method requires serial axial sectioning of arteries and subsequent histopathologic staining. This approach is labor intensive, and its inherent destructiveness undermines the accuracy of lesion-volume determinations. In en face analysis, the aorta is opened longitudinally and stained to reveal lipid-laden plaques; the stained area is quantified from photographs. The speed and ease of en face analysis have made it the most widely used method for measuring atherosclerotic plaques ex vivo , but this method gives only a 2-dimensional (2D) measure of plaque burden. Here, we describe the nonlinear optical 3D (NLO-3D) method. Aortas from euthanized Apoe −/− mice fed a high-fat diet were dissected, stained with oil red O as described,4 and placed in a holder to permit measurement by either NLO-3D imaging or the en face method (Figure IA in the Data Supplement). We used a single excitation wavelength (800 nm) of a commercially available 2-photon instrument to simultaneously generate 3 confocal signals from these aortas (Figure IB in the Data Supplement): second harmonic generation from collagen and 2-photon excitation fluorescence from elastin and from oil red. A Zeiss 780 upright microscope equipped with a ×10 air objective (Zeiss Plan …