Dysfunctional polycomb transcriptional repression contributes to lamin A/C-dependent muscular dystrophy.

Lamin A is a component of the inner nuclear membrane that, together with epigenetic factors, organizes the genome in higher order structures required for transcriptional control. Mutations in the lamin A/C gene cause several diseases belonging to the class of laminopathies, including muscular dystrophies. Nevertheless, molecular mechanisms involved in the pathogenesis of lamin A-dependent dystrophies are still largely unknown. The polycomb group (PcG) of proteins are epigenetic repressors and lamin A interactors, primarily involved in the maintenance of cell identity. Using a murine model of Emery-Dreifuss muscular dystrophy (EDMD), we show here that lamin A loss deregulated PcG positioning in muscle satellite stem cells, leading to derepression of non-muscle-specific genes and p16INK4a, a senescence driver encoded in the Cdkn2a locus. This aberrant transcriptional program caused impairment in self-renewal, loss of cell identity, and premature exhaustion of the quiescent satellite cell pool. Genetic ablation of the Cdkn2a locus restored muscle stem cell properties in lamin A/C-null dystrophic mice. Our findings establish a direct link between lamin A and PcG epigenetic silencing and indicate that lamin A-dependent muscular dystrophy can be ascribed to intrinsic epigenetic dysfunctions of muscle stem cells.

Constitutional abnormality of nuclear membrane proteins in small cell lung carcinoma.

Nuclear membrane proteins reportedly play important roles in maintaining nuclear structures and coordinating cell activities. Studying profiles of nuclear membrane proteins may help us evaluate the biological and/or clinical nature of malignant tumors. Using immunohistochemistry with antibodies for emerin, lamin A/C, lamin B, and LAP2, we examined 105 lung cancer tissues from 33 small cell lung carcinomas (SCLCs) and 72 non-SCLCs (34 adenocarcinomas, 30 squamous cell carcinomas, and 8 large cell carcinomas). Emerin had negative or local/weak positivity in 79% of SCLCs and 1% of non-SCLCs, and lamin A/C had similar positivity in 91% of SCLCs and 3% of non-SCLCs. LAP2's expression was similar between SCLCs and non-SCLCs. RT-PCR analyses for these four nuclear membrane proteins over 7 cell lines showed that mRNA of emerin and lamin A/C were distinctly downregulated in the SCLC cell lines, supporting the immunohistochemical results. In conclusion, we suggest that downregulation of the nuclear membrane proteins emerin and lamin A/C is characteristic of SCLC cells, and this constitutional abnormality of the nuclear membrane may be related to the biological and/or clinical nature of SCLC. In addition, knowing the nuclear protein profile in SCLC cells may contribute to our understanding of nuclear fragility known as the crush artifact in pulmonary biopsy specimens.

Expression of lamin A/C protein in degenerated human intervertebral disc.

OBJECTIVE: This study aimed to evaluate the expression characteristics of lamin A/C proteins in intervertebral disc degeneration (IVD) specimens from patients with different degeneration grades. Lamin A/C proteins have been shown to result in age-related changes in the osteoarticular system. However, the expression characteristics of these nuclear proteins in degenerated human IVD tissues have not been explored previously. PATIENTS AND METHODS: Degenerated human IVD tissues were obtained during spinal surgery. Articular cartilage samples after total knee replacement surgery were used as controls. Sections of these tissues were stained with hematoxylin and eosin, Masson, safranin O, and immunostained using lamin A/C antibody. Western blot was performed to evaluate lamin A/C expression in IVD tissues. Lamin A/C expression was analyzed based on different degeneration grades. RESULTS: In patients with IVD degeneration, mild or moderate degenerative discs contained high amounts of lamin A/C proteins. Lamin A/C expression was primarily localized in the nuclear envelope of IVD cells, and associated with apoptosis in cell nuclei, as determined by immunostaining and TUNEL assay. CONCLUSIONS: This paper is the first to report that lamin A/C proteins are present in IVD tissues and its expression may be related to disc degeneration.

ZMPSTE24 missense mutations that cause progeroid diseases decrease prelamin A cleavage activity and/or protein stability.

The human zinc metalloprotease ZMPSTE24 is an integral membrane protein crucial for the final step in the biogenesis of the nuclear scaffold protein lamin A, encoded by LMNA After farnesylation and carboxyl methylation of its C-terminal CAAX motif, the lamin A precursor (prelamin A) undergoes proteolytic removal of its modified C-terminal 15 amino acids by ZMPSTE24. Mutations in LMNA or ZMPSTE24 that impede this prelamin A cleavage step cause the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS), and the related progeroid disorders mandibuloacral dysplasia type B (MAD-B) and restrictive dermopathy (RD). Here, we report the development of a 'humanized yeast system' to assay ZMPSTE24-dependent cleavage of prelamin A and examine the eight known disease-associated ZMPSTE24 missense mutations. All mutations show diminished prelamin A processing and fall into three classes, with defects in activity, protein stability or both. Notably, some ZMPSTE24 mutants can be rescued by deleting the E3 ubiquitin ligase Doa10, involved in endoplasmic reticulum (ER)-associated degradation of misfolded membrane proteins, or by treatment with the proteasome inhibitor bortezomib. This finding may have important therapeutic implications for some patients. We also show that ZMPSTE24-mediated prelamin A cleavage can be uncoupled from the recently discovered role of ZMPSTE24 in clearance of ER membrane translocon-clogged substrates. Together with the crystal structure of ZMPSTE24, this humanized yeast system can guide structure-function studies to uncover mechanisms of prelamin A cleavage, translocon unclogging, and membrane protein folding and stability.

Suspension state increases reattachment of breast cancer cells by up-regulating lamin A/C.

Extravasation is a rate-limiting step of tumor metastasis, for which adhesion to endothelium of circulating tumor cells (CTCs) is the prerequisite. The suspension state of CTCs undergoing detachment from primary tumor is a persistent biomechanical cue, which potentially regulates the biophysical characteristics and cellular behaviors of tumor cells. In this study, breast tumor cells MDA-MB-231 in suspension culture condition were used to investigate the effect of suspension state on reattachment of CTCs. Our study demonstrated that suspension state significantly increased the adhesion ability of breast tumor cells. In addition, suspension state markedly promoted the formation of stress fibers and focal adhesions and reduced the motility in reattached breast cancer cells. Moreover, lamin A/C was reversibly accumulated at posttranscriptional level under suspension state, improving the cell stiffness of reattached breast cancer cells. Disruption of actin cytoskeleton by cytochalasin D caused lamin A/C accumulation. Conversely, decreasing actomyosin contraction by ROCK inhibitor Y27632 reduced lamin A/C level. Knocking down lamin A/C weakened the suspension-induced increase of adhesion, and also abolished the suspension-induced decrease of motility and increase of stress fibers and focal adhesion in reattaching tumor cells, suggesting a crucial role of lamin A/C. In conclusion, it was demonstrated that suspension state promoted the reattachment of breast tumor cells by up-regulating lamin A/C via cytoskeleton disruption. These findings highlight the important role of suspension state for tumor cells in tumor metastasis.

Causes and consequences of nuclear envelope alterations in tumour progression.

Morphological changes in the size and shape of the nucleus are highly prevalent in cancer, but the underlying molecular mechanisms and the functional relevance remain poorly understood. Nuclear envelope proteins, which can modulate nuclear shape and organization, have emerged as key components in a variety of signalling pathways long implicated in tumourigenesis and metastasis. The expression of nuclear envelope proteins is altered in many cancers, and changes in levels of nuclear envelope proteins lamins A and C are associated with poor prognosis in multiple human cancers. In this review we highlight the role of the nuclear envelope in different processes important for tumour initiation and cancer progression, with a focus on lamins A and C. Lamin A/C controls many cellular processes with key roles in cancer, including cell invasion, stemness, genomic stability, signal transduction, transcriptional regulation, and resistance to mechanical stress. In addition, we discuss potential mechanisms mediating the changes in lamin levels observed in many cancers. A better understanding of cause-and-effect relationships between lamin expression and tumour progression could reveal important mechanisms for coordinated regulation of oncogenic processes, and indicate therapeutic vulnerabilities that could be exploited for improved patient outcome.

Cardiomyocyte-enriched protein CIP protects against pathophysiological stresses and regulates cardiac homeostasis.

Cardiomyopathy is a common human disorder that is characterized by contractile dysfunction and cardiac remodeling. Genetic mutations and altered expression of genes encoding many signaling molecules and contractile proteins are associated with cardiomyopathy; however, how cardiomyocytes sense pathophysiological stresses in order to then modulate cardiac remodeling remains poorly understood. Here, we have described a regulator in the heart that harmonizes the progression of cardiac hypertrophy and dilation. We determined that expression of the myocyte-enriched protein cardiac ISL1-interacting protein (CIP, also known as MLIP) is reduced in patients with dilated cardiomyopathy. As CIP is highly conserved between human and mouse, we evaluated the effects of CIP deficiency on cardiac remodeling in mice. Deletion of the CIP-encoding gene accelerated progress from hypertrophy to heart failure in several cardiomyopathy models. Conversely, transgenic and AAV-mediated CIP overexpression prevented pathologic remodeling and preserved cardiac function. CIP deficiency combined with lamin A/C deletion resulted in severe dilated cardiomyopathy and cardiac dysfunction in the absence of stress. Transcriptome analyses of CIP-deficient hearts revealed that the p53- and FOXO1-mediated gene networks related to homeostasis are disturbed upon pressure overload stress. Moreover, FOXO1 overexpression suppressed stress-induced cardiomyocyte hypertrophy in CIP-deficient cardiomyocytes. Our studies identify CIP as a key regulator of cardiomyopathy that has potential as a therapeutic target to attenuate heart failure progression.

Concentration-dependent Effects of Nuclear Lamins on Nuclear Size in Xenopus and Mammalian Cells.

A fundamental question in cell biology concerns the regulation of organelle size. While nuclear size is exquisitely controlled in different cell types, inappropriate nuclear enlargement is used to diagnose and stage cancer. Clarifying the functional significance of nuclear size necessitates an understanding of the mechanisms and proteins that control nuclear size. One structural component implicated in the regulation of nuclear morphology is the nuclear lamina, a meshwork of intermediate lamin filaments that lines the inner nuclear membrane. However, there has not been a systematic investigation of how the level and type of lamin expression influences nuclear size, in part due to difficulties in precisely controlling lamin expression levels in vivo. In this study, we circumvent this limitation by studying nuclei in Xenopus laevis egg and embryo extracts, open biochemical systems that allow for precise manipulation of lamin levels by the addition of recombinant proteins. We find that nuclear growth and size are sensitive to the levels of nuclear lamins, with low and high concentrations increasing and decreasing nuclear size, respectively. Interestingly, each type of lamin that we tested (lamins B1, B2, B3, and A) similarly affected nuclear size whether added alone or in combination, suggesting that total lamin concentration, and not lamin type, is more critical to determining nuclear size. Furthermore, we show that altering lamin levels in vivo, both in Xenopus embryos and mammalian tissue culture cells, also impacts nuclear size. These results have implications for normal development and carcinogenesis where both nuclear size and lamin expression levels change.

Modulation of TGFbeta 2 levels by lamin A in U2-OS osteoblast-like cells: understanding the osteolytic process triggered by altered lamins.

Transforming growth factor beta (TGFbeta) plays an essential role in bone homeostasis and deregulation of TGFbeta occurs in bone pathologies. Patients affected by Mandibuloacral Dysplasia (MADA), a progeroid disease linked to LMNA mutations, suffer from an osteolytic process. Our previous work showed that MADA osteoblasts secrete excess amount of TGFbeta 2, which in turn elicits differentiation of human blood precursors into osteoclasts. Here, we sought to determine how altered lamin A affects TGFbeta signaling. Our results show that wild-type lamin A negatively modulates TGFbeta 2 levels in osteoblast-like U2-OS cells, while the R527H mutated prelamin A as well as farnesylated prelamin A do not, ultimately leading to increased secretion of TGFbeta 2. TGFbeta 2 in turn, triggers the Akt/mTOR pathway and upregulates osteoprotegerin and cathepsin K. TGFbeta 2 neutralization rescues Akt/mTOR activation and the downstream transcriptional effects, an effect also obtained by statins or RAD001 treatment. Our results unravel an unexpected role of lamin A in TGFbeta 2 regulation and indicate rapamycin analogs and neutralizing antibodies to TGFbeta 2 as new potential therapeutic tools for MADA.

Lamin A/C Acts as an Essential Factor in Mesenchymal Stem Cell Differentiation Through the Regulation of the Dynamics of the Wnt/ß-Catenin Pathway.

Changes in the expression of lamin A/C, a fibrilar protein of the nuclear envelope, are associated with the cellular features of age-related bone loss. Reduced expression of lamin A/C inhibits osteoblastogenesis while facilitating adipogenic differentiation of mesenchymal stem cells (MSC) in vitro and in vivo. In this study we investigated the regulatory role that lamin A/C plays on the essential elements of the Wnt/ß-catenin pathway, which are pivotal in MSC differentiation. Initially, we assessed the effect of lamin A/C gene (LMNA) overexpression on MSC differentiation while compared it to lamin A/C depleted MSC. Osteogenesis and gene expression of osteogenic factors were higher in LMNA-transfected MSC as compared to control. Conversely, adipogenesis and expression of adipogenic factors were significantly lower in LMNA transfected cells. Nuclear ß-catenin was significantly higher (∼two fold) in MSC expressing higher levels of LMNA as compared to control with nuclear ß-catenin levels being significantly lower (∼ -42%) in siRNA-treated MSC. Luciferase activity for ß-catenin-mediated transcriptional activation was significantly higher in cells overexpressing LMNA. These data indicate that MSC overexpressing LMNA have higher osteogenic and lower adipogenic differentiation potential. In conclusion, our studies demonstrate that lamin A/C plays a significant role in the differentiation of both osteoblasts and adipocytes by regulating some of the elements of Wnt/ß-catenin signaling during early MSC differentiation.

The role of miR-205 in the VEGF-mediated promotion of human ovarian cancer cell invasion.

OBJECTIVE: Our objective was to investigate a miRNA pathway that acts downstream of VEGF-induced invasion of ovarian cancer cells. METHOD: We used two paired high and low metastatic serous ovarian cancer cells to demonstrate the role of miR-205 in VEGF-induced invasion of ovarian cancer cells and to investigate the gene targets of miR-205. RESULTS: Our previous comparative proteomics studies showed that VEGF decreased the expression of Ezrin and Lamin A/C, and this result was validated in the present study using qPCR and Western blotting. Then we found that VEGF enhanced the invasiveness of and inhibited apoptosis in ovarian cancer cells as assessed by transwell invasion assays and Annexin V-FITC immunostaining, respectively. VEGFR was also expressed in ovarian cancer cells, as assessed by immunocytochemical staining. Furthermore, using the dual-luciferase report assay system, we demonstrated that miR-205 targeted Ezrin and Lamin A/C. MiR-205 was up-regulated in ovarian cancer cells exposed to VEGF, as determined by miRNA microarray analysis and verified by qPCR. MiR-205 promoted the invasion and proliferation of ovarian cancer cells. CONCLUSION: Our data reveal a new potential pathway in which VEGF promotes the invasion of ovarian cancer cells, partially via the down-regulation of Ezrin and Lamin A/C caused by increased expression of miR-205.

Nuclear envelope composition determines the ability of neutrophil-type cells to passage through micron-scale constrictions.

Neutrophils are characterized by their distinct nuclear shape, which is thought to facilitate the transit of these cells through pore spaces less than one-fifth of their diameter. We used human promyelocytic leukemia (HL-60) cells as a model system to investigate the effect of nuclear shape in whole cell deformability. We probed neutrophil-differentiated HL-60 cells lacking expression of lamin B receptor, which fail to develop lobulated nuclei during granulopoiesis and present an in vitro model for Pelger-Huët anomaly; despite the circular morphology of their nuclei, the cells passed through micron-scale constrictions on similar timescales as scrambled controls. We then investigated the unique nuclear envelope composition of neutrophil-differentiated HL-60 cells, which may also impact their deformability; although lamin A is typically down-regulated during granulopoiesis, we genetically modified HL-60 cells to generate a subpopulation of cells with well defined levels of ectopic lamin A. The lamin A-overexpressing neutrophil-type cells showed similar functional characteristics as the mock controls, but they had an impaired ability to pass through micron-scale constrictions. Our results suggest that levels of lamin A have a marked effect on the ability of neutrophils to passage through micron-scale constrictions, whereas the unusual multilobed shape of the neutrophil nucleus is less essential.

Importance and challenge of making an early diagnosis in LMNA-related muscular dystrophy.

OBJECTIVE: To identify the most useful clinical and histologic markers that facilitate early diagnosis in LMNA-related muscular dystrophy and to assess the usefulness of Western blotting (WB) for lamin A/C. METHODS: We analyzed the clinical and histologic features and WB results of all patients with laminopathies diagnosed in a research-based diagnostic service over 8 years. RESULTS: Although patients with congenital muscular dystrophy (MDCL) (n = 5) and Emery-Dreifuss muscular dystrophy (EDMD) (n = 5) had distinctive early clinical features, the lack of a suggestive clinical phenotype significantly delayed diagnosis in 2 of 3 patients with limb-girdle muscular dystrophy (LGMD) (n = 3). In addition, 6 of 20 muscle biopsy samples were considered nondystrophic, which contributed to delays in diagnosis in some patients. Neck extensor involvement (weakness or contractures) was the most consistent clinical sign, present in all patients. Reduced lamin A/C levels on WB were seen in 5 of 9 patients with laminopathies. CONCLUSION: Clinical features provide the best clues for diagnosing MDCL and EDMD early in the disease, and we urge clinicians to become familiar with those phenotypes. WB for lamin A/C may contribute to diagnosis but requires technical expertise, and results are normal in many individuals with LMNA mutations. Because of the survival benefit of early diagnosis and treatment, we recommend that LMNA gene sequencing be performed in all patients with undiagnosed congenital muscular dystrophy and neck extensor weakness, all patients with genetically undiagnosed LGMD, and those with suggestive clinical signs and nonspecific histologic abnormalities.

Nuclear envelope structural defects cause chromosomal numerical instability and aneuploidy in ovarian cancer.

BACKGROUND: Despite our substantial understanding of molecular mechanisms and gene mutations involved in cancer, the technical approaches for diagnosis and prognosis of cancer are limited. In routine clinical diagnosis of cancer, the procedure is very basic: nuclear morphology is used as a common assessment of the degree of malignancy, and hence acts as a prognostic and predictive indicator of the disease. Furthermore, though the atypical nuclear morphology of cancer cells is believed to be a consequence of oncogenic signaling, the molecular basis remains unclear. Another common characteristic of human cancer is aneuploidy, but the causes and its role in carcinogenesis are not well established. METHODS: We investigated the expression of the nuclear envelope proteins lamin A/C in ovarian cancer by immunohistochemistry and studied the consequence of lamin A/C suppression using siRNA in primary human ovarian surface epithelial cells in culture. We used immunofluorescence microscopy to analyze nuclear morphology, flow cytometry to analyze cellular DNA content, and fluorescence in situ hybridization to examine cell ploidy of the lamin A/C-suppressed cells. RESULTS: We found that nuclear lamina proteins lamin A/C are often absent (47%) in ovarian cancer cells and tissues. Even in lamin A/C-positive ovarian cancer, the expression is heterogeneous within the population of tumor cells. In most cancer cell lines, a significant fraction of the lamin A/C-negative population was observed to intermix with the lamin A/C-positive cells. Down regulation of lamin A/C in non-cancerous primary ovarian surface epithelial cells led to morphological deformation and development of aneuploidy. The aneuploid cells became growth retarded due to a p53-dependent induction of the cell cycle inhibitor p21. CONCLUSIONS: We conclude that the loss of nuclear envelope structural proteins, such as lamin A/C, may underlie two of the hallmarks of cancer--aberrations in nuclear morphology and aneuploidy.

Genes involved in cell adhesion, cell motility and mitogenic signaling are altered due to HPV 16 E5 protein expression.

We investigated the effects of the human papillomavirus type 16 E5 oncogene on cellular gene expression in human epithelial cells using cDNA microarray. In a genome-wide microarray assay, the expression of 179 genes was found to be significantly altered due to E5 expression. The expression of lamin A/C was downregulated at protein level. The expression of protein kinase C-delta and phosphoinositide-3-kinase proteins was found to be upregulated. We also observed increased motility of E5-expressing cells. We conclude that the E5 protein affects several cellular pathways involved in cell adhesion, cell motility and mitogenic signaling. These alterations may together lead to inhibition of apoptosis and facilitate the establishment of persistent infection in the epithelium.

Age-related changes in lamin A/C expression in the osteoarticular system: laminopathies as a potential new aging mechanism.

BACKGROUND: Mutations in lamin A/C have been described as associated to severe changes in bone and joints. In this study we hypothesize that the expression of lamin A/C may play a role in the pathogenesis of age-related diseases in the osteoarticular system. METHODOLOGY: C57BL/6 young and old mice (4 months; n=10 and 24 months; n=10) were sacrificed. Limbs were isolated for histopathological and Western blot analysis. The proportion of cells (osteoblasts and chondrocytes) positive for lamin A/C was quantified by immunohistochemistry. Lamin B1 was used as control. Finally, lamin A/C expression in bone marrow cells was quantified by Western blot. RESULTS: A significant reduction in lamin A/C was found in osteoblasts of old as compared to young mice (42% versus 76%, p<0.001). Interestingly, lamin A/C but not lamin B1 expression was found in bone matrix with higher levels in young bone. Additionally, a significant reduction in the number of lamin A/C expressing chondrocytes was seen in old mice as compared to young mice (32% versus 84%, p<0.001). Finally, a reduction in lamin A/C expression was found in bone marrow cells obtained from old mice as compared to young mice. CONCLUSION: This is the first assessment of the age-related changes in lamin A/C expression in the osteoarticular system. We conclude that with aging there is a reduction in lamin A/C expression which could have a significance on osteoarticular cells function and viability.

Lamin A/C-dependent localization of Nesprin-2, a giant scaffolder at the nuclear envelope.

The vertebrate proteins Nesprin-1 and Nesprin-2 (also referred to as Enaptin and NUANCE) together with ANC-1 of Caenorhabditis elegans and MSP-300 of Drosophila melanogaster belong to a novel family of alpha-actinin type actin-binding proteins residing at the nuclear membrane. Using biochemical techniques, we demonstrate that Nesprin-2 binds directly to emerin and the C-terminal common region of lamin A/C. Selective disruption of the lamin A/C network in COS7 cells, using a dominant negative lamin B mutant, resulted in the redistribution of Nesprin-2. Furthermore, using lamin A/C knockout fibroblasts we show that lamin A/C is necessary for the nuclear envelope localization of Nesprin-2. In normal skin where lamin A/C is differentially expressed, strong Nesprin-2 expression was found in all epidermal layers, including the basal layer where only lamin C is present. This indicates that lamin C is sufficient for proper Nesprin-2 localization at the nuclear envelope. Expression of dominant negative Nesprin-2 constructs and knockdown studies in COS7 cells revealed that the presence of Nesprin-2 at the nuclear envelope is necessary for the proper localization of emerin. Our data imply a scaffolding function of Nesprin-2 at the nuclear membrane and suggest a potential involvement of this multi-isomeric protein in human disease.

p300 modulates nuclear morphology in prostate cancer.

Alterations in nuclear structure distinguish cancer cells from noncancer cells. These nuclear alterations can be translated into quantifiable features by digital image analysis in a process known as quantitative nuclear morphometry. Recently, quantitative nuclear morphometry has been shown to predict metastasis and biochemical recurrence of prostate cancer. However, little is known about the cellular mechanisms underlying these nuclear morphometric changes. Alterations of nuclear matrix proteins are frequently involved in changes of nuclear structure. A number of co-activators interact with these nuclear structure-related proteins, suggesting that they might be involved in quantitative nuclear morphometry changes. We have shown previously that the transcriptional co-activator p300 is involved in prostate cancer progression. However, the ability of a transcriptional regulator like p300 to modulate nuclear morphology has not been described previously. In the present study, we show that p300 expression in prostate cancer biopsy tissue from 95 patients correlates with quantifiable nuclear alterations. Moreover, we show that transfection of p300 into prostate cancer cells in culture induces quantifiable nuclear alterations, such as diameter, perimeter, and absorbance among others, as assessed by digital image analysis. These alterations correlate individually with aggressive features in prostate cancer, such as expression of the proliferation marker Ki-67 and extraprostatic extension of the tumor. Finally, we found that transfection of p300 into prostate cancer cells specifically increases mRNA and protein levels of nuclear matrix peptides lamins A and C, suggesting that these proteins mediate the p300-induced effects. These findings reveal a new insight into the transcriptional and structural regulation of prostate cancer.