Novel Nuclear Nesprin-2 Variants Tether Active Extracellular Signal-regulated MAPK1 and MAPK2 at Promyelocytic Leukemia Protein Nuclear Bodies and Act to Regulate Smooth Muscle Cell Proliferation

Nuclear and cytoplasmic scaffold proteins have been shown to be essential for temporal and spatial organization, as well as the fidelity, of MAPK signaling pathways. In this study we show that nesprin-2 is a novel extracellular signal-regulated MAPK1 and 2 (ERK1/2) scaffold protein that serves to regulate nuclear signaling by tethering these kinases at promyelocytic leukemia protein nuclear bodies (PML NBs). Using immunofluorescence microscopy, GST pull-down and immunoprecipitation, we show that nesprin-2, ERK1/2, and PML colocalize and bind to form a nuclear complex. Interference of nesprin-2 function, by either siRNA-mediated knockdown or overexpression of a dominant negative nesprin-2 fragment, augmented ERK1/2 nuclear signaling shown by increased SP1 activity and ELK1 phosphorylation. The functional outcome of nesprin-2 disruption and the resultant sustained ERK1/2 signal was increased proliferation. Importantly, these activities were not induced by previously identified nuclear envelope (NE)-targeted nesprin-2 isoforms but rather were mediated by novel nuclear isoforms that lacked the KASH domain. Taken together, this study suggests that nesprin-2 is a novel intranuclear scaffold, essential for nuclear ERK1/2 signaling fidelity and cell cycle progression.     

Distinct functional domains in nesprin-1alpha and nesprin-2beta bind directly to emerin and both interactions are disrupted in X-linked Emery-Dreifuss muscular dystrophy

Emerin and specific isoforms of nesprin-1 and -2 are nuclear membrane proteins which are binding partners in multi-protein complexes spanning the nuclear envelope. We report here the characterisation of the residues both in emerin and in nesprin-1alpha and -2beta which are involved in their interaction and show that emerin requires nesprin-1 or -2 to retain it at the nuclear membrane. Using several protein-protein interaction methods, we show that residues 368 to 627 of nesprin-1alpha and residues 126 to 219 of nesprin-2beta, which show high homology to one another, both mediate binding to emerin residues 140-176. This region has previously been implicated in binding to F-actin, beta-catenin and lamin A/C suggesting that it is critical for emerin function. Confirmation that these protein domains interact in vivo was shown using GFP-dominant negative assays. Exogenous expression of either of these nesprin fragments in mouse myoblast C2C12 cells displaced endogenous emerin from the nuclear envelope and reduced the targeting of newly synthesised emerin. Furthermore, we are the first to report that emerin mutations which give rise to X-linked Emery-Dreifuss muscular dystrophy, disrupt binding to both nesprin-1alpha and -2beta isoforms, further indicating a role of nesprins in the pathology of Emery-Dreifuss muscular dystrophy.     

Increase of brain-derived neurotrophic factor gene expression in NG108-15 cells by the nuclear isoforms of Ca2+/ calmodulin-dependent protein kinase II

We have reported that the delta3 isoform of Ca2+/ calmodulin-dependent protein kinase II (CaM kinase II) is abundant in the nucleus in cerebellar granule cells. To examine the possibility that the nuclear isoforms of CaM kinase II are involved in the expression of brain-derived neurotrophic factor (BDNF), we transiently overexpressed the delta3 isoform in NG108-15 cells. The quantitative RT-PCR analysis revealed that rat cerebellum and NG108-15 cells expressed the exon IV-containing mRNA of BDNF (exon IV-BDNF mRNA) more than the exon III-BDNF mRNA. Treatment of NG108-15 cells with Bay K 8644 increased both exon III- and exon IV-BDNF mRNAs, and overexpression of the 83 isoform potentiated the expression of the exon IV-BDNF mRNA. The potentiation was not observed in the cells that were overexpressed with either the 61 isoform, a nonnuclear isoform, or the inactive mutant of the delta3 isoform. We constructed the luciferase reporter gene following the promoter upstream of exon IV and confirmed that overexpression of the delta3 isoform increased luciferase gene expression. Double-immunostaining of NG108-15 cells with the antibodies to CaM kinase II and BDNF clearly showed that BDNF was highly expressed in the cells that were overexpressed with the delta3 isoform or the alphaB isoform, another nuclear isoform of CaM kinase II. These results suggest that the nuclear isoforms of CaM kinase II are involved in the expression of BDNF.     

Activation of nuclear Ca(2+)/calmodulin-dependent protein kinase II and brain-derived neurotrophic factor gene expression by stimulation of dopamine D2 receptor in transfected NG108-15 cells

We identified the isoforms of Ca(2+) /calmodulin-dependent protein kinase II (CaM kinase II) subunits in rat striatum. All four subunits of CaM kinase II alpha, beta, gamma and delta were detected including the isoforms of alphaB, gammaA, gammaA', gammaA.B, delta3 and delta7 with nuclear localization signal. We established NG108-15 cells with the stably expressed dopamine D2L receptor (D2LR, long form), which is an alternative splicing variant. The cells were termed NGD2L. Immunostaining demonstrated that D2LR was localized in plasma membranes. Calcium imaging with fluo-3 AM revealed that quinpirole, a D2R agonist, increased the intracellular Ca(2+), which was blocked by treatment with sulpiride and pertussis toxin in NGD2L cells, but not in mock cells. Furthermore, stimulation of D2LR with quinpirole in NGD2L cells activated the nuclear isoform of CaM kinase II. Stimulation of D2LR increased the expression of exon III- and IV-BDNF mRNA. Overexpression of CaM kinase II delta3 increased exon IV- but not exon III-BDNF mRNA. These results suggest that D2R is involved in the activation of the nuclear isoform of CaM kinase II and thereby in stimulation of gene expression through Ca(2+) signaling.     

Nesprin-1alpha contributes to the targeting of mAKAP to the cardiac myocyte nuclear envelope

Muscle A-kinase anchoring protein (mAKAP) is a scaffold protein found principally at the nuclear envelope of striated myocytes. mAKAP maintains a complex consisting of multiple signal transduction molecules including the cAMP-dependent protein kinase A, the ryanodine receptor calcium release channel, phosphodiesterase type 4D3, and protein phosphatase 2A. By an unknown mechanism, a domain containing spectrin repeats is responsible for targeting mAKAP to the nuclear envelope. We now demonstrate that the integral membrane protein nesprin-1alpha serves as a receptor for mAKAP on the nuclear envelope in cardiac myocytes. Nesprin-1alpha is inserted into the nuclear envelope by a conserved, C-terminal, klarsicht-related transmembrane domain and forms homodimers by the binding of an amino-terminal spectrin repeat domain. Through the direct binding of the nesprin-1alpha amino-terminal dimerization domain to the third mAKAP spectrin repeat, nesprin-1alpha targets mAKAP to the nuclear envelope. In turn, overexpression of these spectrin repeat domains in myocytes can displace mAKAP from nesprin-1alpha.     

Dystonin/Bpag1 is a necessary endoplasmic reticulum/nuclear envelope protein in sensory neurons

Dystonin/Bpag1 proteins are cytoskeletal linkers whose loss of function in mice results in a hereditary sensory neuropathy with a progressive loss of limb coordination starting in the second week of life. These mice, named dystonia musculorum (dt), succumb to the disease and die of unknown causes prior to sexual maturity. Previous evidence indicated that cytoskeletal defects in the axon are a primary cause of dt neurodegeneration. However, more recent data suggests that other factors may be equally important contributors to the disease process. In the present study, we demonstrate perikaryal defects in dorsal root ganglion (DRG) neurons at stages preceding the onset of loss of limb coordination in dt mice. Abnormalities include alterations in endoplasmic reticulum (ER) chaperone protein expression, indicative of an ER stress response. Dystonin in sensory neurons localized in association with the ER and nuclear envelope (NE). A fusion protein ofthe dystonin-a2 isoform, which harbors an N-terminal transmembrane domain, associated with and reorganized the ER in cell culture. This isoform also interacts with the NE protein nesprin-3α, but not nesprin-3β. Defects in dt mice, as demonstrated here, may ultimately result in pathogenesis involving ER dysfunction and contribute significantly to the dt phenotype.     

CDK2 differentially controls normal cell senescence and cancer cell proliferation upon exposure to reactive oxygen species

The linker of nucleus and cytoskeleton (LINC) complex, including nesprin-1, has been suggested to be crucial for many biological processes. Previous studies have shown that mutations in nesprin-1 cause abnormal cellular functions and diseases, possibly because of insufficient force transmission to the nucleus through actin filaments (F-actin) bound to nesprin-1. However, little is known regarding the mechanical interaction between the nucleus and F-actin through nesprin-1. In this study, we examined nuclear deformation behavior in nesprin-1 knocked-down endothelial cells (ECs) subjected to uniaxial stretching by evaluating nuclear strain from lateral cross-sectional images. The widths of nuclei in nesprin-1 knocked-down ECs were smaller than those in wild-type cells. In addition, nuclear strain in nesprin-1 knocked-down cells, which is considered to be compressed by the actin cortical layer, increased compared with that in wild-type cells under stretching condition. These results indicate that nesprin-1 knockdown releases the nucleus from the tension of F-actin bound to the nucleus, thereby increasing allowance for deformation before stretching, and that F-actin bound to the nucleus through nesprin-1 causes sustainable force transmission to the nucleus.     

A Highlights from MBoC Selection: Nesprin-3 connects plectin and vimentin to the nuclear envelope of Sertoli cells but is not required for Sertoli cell function in spermatogenesis

Nesprin-3 is a nuclear envelope protein that connects the nucleus to intermediate filaments by interacting with plectin. To investigate the role of nesprin-3 in the perinuclear localization of plectin, we generated nesprin-3–knockout mice and examined the effects of nesprin-3 deficiency in different cell types and tissues. Nesprin-3 and plectin are coexpressed in a variety of tissues, including peripheral nerve and muscle. The expression level of nesprin-3 in skeletal muscle is very low and decreases during myoblast differentiation in vitro. Of interest, plectin was concentrated at the nuclear envelope in only a few cell types. This was most prominent in Sertoli cells of the testis, in which nesprin-3 is required for the localization of both plectin and vimentin at the nuclear perimeter. Testicular morphology and the position of the nucleus in Sertoli cells were normal, however, in the nesprin-3–knockout mice and the mice were fertile. Furthermore, nesprin-3 was not required for the polarization and migration of mouse embryonic fibroblasts. Thus, although nesprin-3 is critical for the localization of plectin to the nuclear perimeter of Sertoli cells, the resulting link between the nuclear envelope and the intermediate filament system seems to be dispensable for normal testicular morphology and spermatogenesis.     

Nesprin-3: a versatile connector between the nucleus and the cytoskeleton

The cytoskeleton is connected to the nuclear interior by LINC (linker of nucleoskeleton and cytoskeleton) complexes located in the nuclear envelope. These complexes consist of SUN proteins and nesprins present in the inner and outer nuclear membrane respectively. Whereas SUN proteins can bind the nuclear lamina, members of the nesprin protein family connect the nucleus to different components of the cytoskeleton. Nesprin-1 and -2 can establish a direct link with actin filaments, whereas nesprin-4 associates indirectly with microtubules through its interaction with kinesin-1. Nesprin-3 is the only family member known that can link the nuclear envelope to intermediate filaments. This indirect interaction is mediated by the binding of nesprin-3 to the cytoskeletal linker protein plectin. Furthermore, nesprin-3 can connect the nucleus to microtubules by its interactions with BPAG1 (bullous pemphigoid antigen 1) and MACF (microtubule-actin cross-linking factor). In contrast with the active roles that nesprin-1, -2 and -4 have in actin- and microtubule-dependent nuclear positioning, the role of nesprin-3 is likely to be more passive. We suggest that it helps to stabilize the anchorage of the nucleus within the cytoplasm and maintain the structural integrity and shape of the nucleus.     

Control of CYP11B2 gene expression through differential regulation of its promoter by atypical and conventional protein kinase C isoforms

We reported previously that the protein kinase C (PKC) inhibitor GF109203X stimulated the hamster CYP11B2 promoter activity in transfected NCI-H295 cells. PKCalpha, -epsilon, and -zeta were detected in hamster adrenal zona glomerulosa and NCI-H295 cells, and PKCtheta in NCI-H295 cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) inhibited basal and stimulated cytochrome P450 aldosterone synthase mRNA expression by angiotensin (AII), dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP), or KCl in NCI-H295 cells. Basal CYP11B2 promoter activity was inhibited in cells cotransfected with constitutively active (CA) PKCalpha, -epsilon, and -theta mutants, whereas it was increased with CA-PKCzeta. Dominant negative (DN) PKCalpha, -theta, -epsilon, and -zeta mutants stimulated the promoter activity. AII-, KCl-, and Bt2cAMP-stimulatory effects were abolished in cells cotransfected with CA-PKCalpha, -epsilon, or -theta. The effect of Bt2cAMP was abolished by CA-PKCzeta but AII and KCl were still able to enhance the promoter activity. DN-PKCalpha, -epsilon, -theta, or -zeta did not inhibit these effects. G?6976 enhanced promoter activity, providing further evidence that PKCalpha was involved. Various CYP11B2 promoter constructs were used to identify the area associated with TPA and PKC inhibition. TPA and CA-PKCalpha, -epsilon, or -theta abolished the effects of AII, KCl, and Bt2cAMP on the activity of -102 and longer constructs. In summary, our findings suggest that the hamster CYP11B2 gene is under differential control by conventional (alpha) and atypical (zeta) PKC.     

梅山猪胚胎附植期EphB2的组织表达及RNA-seq分析

猪产仔数是一个重要的经济和繁殖性状, 而胚胎附植是影响猪产仔数的重要因素。为了研究促红细胞生成素产生肝细胞受体B2 (EphB2)对猪胚胎附植过程中子宫内膜迁移和粘附活动的影响, 文章以太湖流域梅山猪为研究对象, 利用实时荧光定量PCR (qRT-PCR)和蛋白免疫印迹(Western blot)方法, 检测EphB2基因在梅山猪胚胎附植前、中、后期子宫内膜附植点/非附植点和卵巢组织的mRNA和蛋白表达谱, 并用转录组测序(RNA-seq)方法分析了不同附植时期子宫内膜附植点和卵巢组织的差异表达基因。qRT-PCR和Western blot结果表明, EphB2在胚胎附植前、中、后期子宫内膜附植点和非附植点的mRNA和蛋白均呈现先升高后降低的表达趋势, 且附植中期的表达量显著高于前期和后期(P<0.01);EphB2在附植前、中、后期卵巢中的mRNA和蛋白表达趋势则相反, 为先降低后升高, 且不同时期间表达差异显著(P<0.05)。RNA-seq结果表明, EphB2在子宫内膜附植点的mRNA表达, 附植中期极显著高于附植前期(P<0.01);EphB2在卵巢的mRNA表达, 附植中期显著高于附植后期(P<0.05)。综上所述, EphB2很可能在猪胚胎附植过程中发挥着重要的调控作用, 为潜在的猪产仔数性状候选基因。     

Regulation of Dab2 expression in intestinal and renal epithelia by development

Disabled-2 (Dab2) is an intracellular adaptor protein proposed to function in endocytosis. Here, we investigate the intestinal and renal Dab2 expression versus maturation. Dab2 mRNA levels measured by RT-PCR are greater in the small than in the large intestine. Immunological studies localize Dab2 to the terminal web domain of the enterocytes and reveal the presence of a 96-kDa Dab2 isoform in the apical membrane of the jejunum, ileum, and renal cortex of the suckling and adult rat. A 69-kDa Dab2 isoform is only observed in the apical membranes of the suckling ileum. During the suckling period, the Dab2 mRNA levels measured in the enterocytes and crypts and those of the 96-kDa Dab2 isoform are greater in the ileum than in the jejunum. No segmental differences are observed in the adult intestine. In the intestine, the levels of Dab2 mRNA and those of the 96-kDa Dab2 isoform decrease to adult values at weaning, whereas in the kidney they increase with development. Weaning the pups on a commercial milk diet slows the periweaning decline in the levels of Dab2 mRNA in the crypts and of those of the 96-kDa isoform. This is the first report showing that the 96-kDa Dab2 isoform is expressed at the apical domain of rat small intestine, that ontogeny regulates Dab2 gene expression in intestine and kidney and that retarding weaning affects intestinal Dab2 gene expression.     

Actomyosin Tension Exerted on the Nucleus through Nesprin-1 Connections Influences Endothelial Cell Adhesion, Migration, and Cyclic Strain-Induced Reorientation

Endothelial cell polarization and directional migration is required for angiogenesis. Polarization and motility requires not only local cytoskeletal remodeling but also the motion of intracellular organelles such as the nucleus. However, the physiological significance of nuclear positioning in the endothelial cell has remained largely unexplored. Here, we show that siRNA knockdown of nesprin-1, a protein present in the linker of nucleus to cytoskeleton complex, abolished the reorientation of endothelial cells in response to cyclic strain. Confocal imaging revealed that the nuclear height is substantially increased in nesprin-1 depleted cells, similar to myosin inhibited cells. Nesprin-1 depletion increased the number of focal adhesions and substrate traction while decreasing the speed of cell migration; however, there was no detectable change in nonmuscle myosin II activity in nesprin-1 deficient cells. Together, these results are consistent with a model in which the nucleus balances a portion of the actomyosin tension in the cell. In the absence of nesprin-1, actomyosin tension is balanced by the substrate, leading to abnormal adhesion, migration, and cyclic strain-induced reorientation.