In many cell types, nuclear A-type lamins have been implicated in structural and functional activities, including higher-order genome organization, DNA replication and repair, gene transcription, and signal transduction. However, their role in specialized immune cells remains largely unexplored. Here, we showed that the abundance of A-type lamins is almost negligible in resting naïve T lymphocytes, but that it is substantially increased upon activation of the T cell receptor (TCR), and is an early event that accelerates formation of the immunological synapse between T cells and antigen-presenting cells. We found that lamin-A enhanced the polymerization of F-actin in T cells, a critical step for immunological synapse formation, by physically connecting the nucleus to the plasma membrane through the linker of nucleoskeleton and cytoskeleton (LINC) complex. We also showed that lamin-A played a key role in other membrane, cytoplasmic, and nuclear events related to TCR activation, including receptor-clustering, downstream signaling, and target gene expression. Notably, the presence of lamin-A was associated with enhanced extracellular signal–regulated kinase 1/2 signaling, and pharmacological inhibition of this pathway reduced the extent of lamin-A–dependent T cell activation. Moreover, mice deficient in lamin-A exhibited impaired T cell responses in vivo. These findings underscore the importance of A-type lamins for TCR activation, and identify lamin-A as a previously unappreciated regulator of the immune response.
Nuclear lamins are important structural and functional proteins in mammalian cells, but little is known about the mechanisms and cofactors that regulate their traffic into the nucleus. Here, we demonstrate that trafficking of lamin A, but not lamin B1, and its assembly into the nuclear envelope are regulated by sorting nexin 6 (SNX6), a major component of the retromer that targets proteins and other molecules to specific subcellular locations. SNX6 interacts with lamin A in vitro and in vivo and links it to the outer surface of the endoplasmic reticulum in human and mouse cells. SNX6 transports its lamin A cargo to the nuclear envelope in a process that takes several hours. Lamin A protein levels in the nucleus augment or decrease, respectively, upon gain or loss of SNX6 function. We further show that SNX6-dependent lamin A nuclear import occurs across the nuclear pore complex via a RAN-GTP-dependent mechanism. These results identify SNX6 as a key regulator of lamin A synthesis and incorporation into the nuclear envelope.
Polμ is an error-prone PolX polymerase that contributes to classical NHEJ DNA repair. Mice lacking Polμ (Polμ−/−) show altered hematopoiesis homeostasis and DSB repair and a more pronounced nucleolytic resection of some V(D)J junctions. We previously showed that Polμ−/− mice have increased learning capacity at old ages, suggesting delayed brain aging. Here we investigated the effect of Polμ−/− deficiency on liver aging. We found that old Polμ−/− mice (>20 month) have greater liver regenerative capacity compared with wt animals. Old Polμ−/− liver showed reduced genomic instability and increased apoptosis resistance. However, Polμ−/− mice did not show an extended life span and other organs (e.g., heart) aged normally. Our results suggest that Polμ deficiency activates transcriptional networks that reduce constitutive apoptosis, leading to enhanced liver repair at old age.
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.
atherosclerosis; hypercholesterolemia; inflammation; macrophage; RCAN1
Atherosclerosis is an inflammatory disease regulated by infiltrating monocytes and T cells, among other cell types. Macrophage recruitment to atherosclerotic lesions is controlled by monocyte infiltration into plaques. Once in the lesion, macrophage proliferation in situ, apoptosis, and differentiation to an inflammatory (M1) or anti-inflammatory phenotype (M2) are involved in progression to advanced atherosclerotic lesions. We studied the role of phosphoinositol-3-kinase (PI3K) p110γ in the regulation of in situ apoptosis, macrophage proliferation and polarization towards M1 or M2 phenotypes in atherosclerotic lesions. We analyzed atherosclerosis development in LDLR−/−p110γ+/− and LDLR−/−p110γ−/− mice, and performed expression and functional assays in tissues and primary cells from these and from p110γ+/− and p110γ−/− mice. Lack of p110γ in LDLR−/− mice reduces the atherosclerosis burden. Atherosclerotic lesions in fat-fed LDLR−/−p110γ−/− mice were smaller than in LDLR−/−p110γ+/− controls, which coincided with decreased macrophage proliferation in LDLR−/−p110γ−/− mouse lesions. This proliferation defect was also observed in p110γ−/− bone marrow-derived macrophages (BMM) stimulated with macrophage colony-stimulating factor (M-CSF), and was associated with higher intracellular cyclic adenosine monophosphate (cAMP) levels. In contrast, T cell proliferation was unaffected in LDLR−/−p110γ−/− mice. Moreover, p110γ deficiency did not affect macrophage polarization towards the M1 or M2 phenotypes or apoptosis in atherosclerotic plaques, or polarization in cultured BMM. Our results suggest that higher cAMP levels and the ensuing inhibition of macrophage proliferation contribute to atheroprotection in LDLR−/− mice lacking p110γ. Nonetheless, p110γ deletion does not appear to be involved in apoptosis, in macrophage polarization or in T cell proliferation.
Transcription factors of the MYC family regulate several homeostatic cell functions, and their role as proto-oncogenes has been the focus of interest for decades. We have recently demonstrated that c-MYC is expressed by tumor-associated macrophages (TAMs) and regulates their phenotype and pro-tumor activities in vivo.
angiogenesis; c-MYC; cancer; macrophage differentiation/maturation; tumor-associated macrophage
Although tumor-associated macrophages (TAMs) are involved in tumor growth and metastasis, the mechanisms controlling their pro-tumoral activities remain largely unknown. The transcription factor c-MYC has been recently shown to regulate in vitro human macrophage polarization and be expressed in macrophages infiltrating human tumors. In this study, we exploited the predominant expression of LysM in myeloid cells to generate c-Mycfl/fl LysMcre/+ mice, which lack c-Myc in macrophages, to investigate the role of macrophage c-MYC expression in cancer. Under steady-state conditions, immune system parameters in c-Mycfl/fl LysMcre/+ mice appeared normal, including the abundance of different subsets of bone marrow hematopoietic stem cells, precursors and circulating cells, macrophage density, and immune organ structure. In a model of melanoma, however, TAMs lacking c-Myc displayed a delay in maturation and showed an attenuation of pro-tumoral functions (e.g., reduced expression of VEGF, MMP9, and HIF1α) that was associated with impaired tissue remodeling and angiogenesis and limited tumor growth in c-Mycfl/fl LysMcre/+ mice. Macrophage c-Myc deletion also diminished fibrosarcoma growth. These data identify c-Myc as a positive regulator of the pro-tumoral program of TAMs and suggest c-Myc inactivation as an attractive target for anti-cancer therapy.
The mechanisms underlying the increased risk of cardiovascular disease associated with diabetes mellitus (DM) are not fully defined. Insulin resistance in human metabolic syndrome patients is associated with decreased expression of the insulin receptor substrate-2- (Irs2-) AKT2 axis in mononuclear leukocytes (MLs). Moreover, acute coronary syndrome (ACS) has been linked through genome-wide association studies to the 2q36-q37.3 locus, which contains the Irs1 gene. Here, we investigated the expression of insulin-signaling pathway genes in MLs from patients with DM, ACS, and ACS plus DM. Quantitative real-time PCR expression studies showed no differences in the mRNA levels of Irs2, Akt2, and Akt1 among all patients. However, Irs1 mRNA expression was significantly increased in patients with ACS—diabetics and nondiabetics—compared with diabetic patients without ACS (P < .02 and P < .005, resp.). The present study reveals for the first time an association between increased Irs1 mRNA levels in MLs of patients with ACS which is not related to DM.
When in the nucleus, ERK1/2 dislodges the retinoblastoma protein from lamin A, facilitating its rapid phosphorylation.
As orchestrators of essential cellular processes like proliferation, ERK1/2 mitogen-activated protein kinase signals impact on cell cycle regulation. A-type lamins are major constituents of the nuclear matrix that also control the cell cycle machinery by largely unknown mechanisms. In this paper, we disclose a functional liaison between ERK1/2 and lamin A whereby cell cycle progression is regulated. We demonstrate that lamin A serves as a mutually exclusive dock for ERK1/2 and the retinoblastoma (Rb) protein. Our results reveal that, immediately after their postactivation entrance in the nucleus, ERK1/2 dislodge Rb from its interaction with lamin A, thereby facilitating its rapid phosphorylation and consequently promoting E2F activation and cell cycle entry. Interestingly, these effects are independent of ERK1/2 kinase activity. We also show that cellular transformation and tumor cell proliferation are dependent on the balance between lamin A and nuclear ERK1/2 levels, which determines Rb accessibility for phosphorylation/inactivation.
A-type lamins (lamins A and C), encoded by the LMNA gene, are major protein constituents of the mammalian nuclear lamina, a complex structure that acts as a scaffold for protein complexes that regulate nuclear structure and functions. Interest in these proteins has increased in recent years with the discovery that LMNA mutations cause a variety of human diseases termed laminopathies, including progeroid syndromes and disorders that primarily affect striated muscle, adipose, bone, and neuronal tissues. In this review, we discuss recent research supporting the concept that lamin A/C and associated nuclear envelope proteins regulate gene expression in health and disease through interplay with signal transduction pathways, transcription factors, and chromatin-associated proteins.
Sequestration of c-Fos at the nuclear envelope (NE) through interaction with A-type lamins suppresses AP-1–dependent transcription. We show here that c-Fos accumulation within the extraction-resistant nuclear fraction (ERNF) and its interaction with lamin A are reduced and enhanced by gain-of and loss-of ERK1/2 activity, respectively. Moreover, hindering ERK1/2-dependent phosphorylation of c-Fos attenuates its release from the ERNF induced by serum and promotes its interaction with lamin A. Accordingly, serum stimulation rapidly releases preexisting c-Fos from the NE via ERK1/2-dependent phosphorylation, leading to a fast activation of AP-1 before de novo c-Fos synthesis. Moreover, lamin A–null cells exhibit increased AP-1 activity and reduced levels of c-Fos phosphorylation. We also find that active ERK1/2 interacts with lamin A and colocalizes with c-Fos and A-type lamins at the NE. Thus, NE-bound ERK1/2 functions as a molecular switch for rapid mitogen-dependent AP-1 activation through phosphorylation-induced release of preexisting c-Fos from its inhibitory interaction with lamin A/C.
Excessive proliferation of vascular smooth muscle cells and leukocytes within the artery wall is a major event in the development of atherosclerosis. The growth suppressor p27kip1 associates with several cyclin-dependent kinase/cyclin complexes, thereby abrogating their capacity to induce progression through the cell cycle. Recent studies have implicated p27kip1 in the control of neointimal hyperplasia. For instance, p27kip1 ablation in apolipoprotein-E-null mice enhanced arterial cell proliferation and accelerated atherogenesis induced by dietary cholesterol. Therefore, p27kip1 is a candidate gene to modify the risk of developing atherosclerosis and associated ischaemic events (i.e., myocardial infarction and stroke).
In this study we found three common single-nucleotide polymorphisms in the human p27kip1 gene (+326T>G [V109G], -79C>T, and -838C>A). The frequency of -838A carriers was significantly increased in myocardial infarction patients compared to healthy controls (odds ratio [OR] = 1.73, 95% confidence interval [95%CI] = 1.12–2.70). In addition, luciferase reporter constructs driven by the human p27kip1 gene promoter containing A at position -838 had decreased basal transcriptional activity when transiently transfected in Jurkat cells, compared with constructs bearing C in -838 (P = 0.04).
These data suggest that -838A is associated with reduced p27kip1 promoter activity and increased risk of myocardial infarction.
myocardial infarction; p27kip1; single-nucleotide polymorphisms
Tight control of cellular growth is essential to ensure normal tissue patterning and prevent pathological responses. Excessive vascular smooth muscle cell (VSMC) proliferation is associated with the pathophysiology of atherosclerosis and restenosis post-angioplasty. Thus, drug targeting of pathological VSMC growth may be a suitable therapeutic intervention in vascular proliferative diseases.In the present study, we investigated the mechanisms underlying VSMC growth arrest induced by the pharmacological agent PCA-4230. Addition of PCA-4230 to cultured VSMCs blocked the induction of cyclin D1 and cyclin A expression normally seen in serum-restimulated cells. Moreover, PCA-4230 inhibited cyclin-dependent kinase 2 (CDK2) activity and abrogated hyperphosphorylation of the retinoblastoma (Rb) gene product. Similarly, PCA-4230-dependent growth arrest of transformed cell lines correlated with reduced level of cyclin D1 protein and inhibition of CDK2 activity. Consistent with these findings, PCA-4230 repressed serum-inducible cyclin A promoter activity, and overexpression of either cyclin D1 or E2F1 efficiently circumvented this inhibitory effect. Importantly, adenovirus-mediated overexpression of E2F1 restored S-phase entry in PCA-4230-treated VSMCs, demonstrating that PCA-4230 represses cyclin A gene expression and VSMC growth via inhibition of the cyclin D1/E2F pathway.Because of its ability to inhibit the growth of human VSMCs and transformed cell lines, future studies are warranted to assess whether PCA-4230 may be a suitable therapeutic intervention for the treatment of hyperproliferative disorders, including cardiovascular disease and cancer.
PCA-4230; 1,4-dihydropyridine; vascular smooth muscle cells; tumour cells; cell cycle