Deleterious assembly of the lamin A/C mutant p.S143P causes ER stress in familial dilated cardiomyopathy.

Mutation of the LMNA gene, encoding nuclear lamin A and lamin C (hereafter lamin A/C), is a common cause of familial dilated cardiomyopathy (DCM). Among Finnish DCM patients, the founder mutation c.427T>C (p.S143P) is the most frequently reported genetic variant. Here, we show that p.S143P lamin A/C is more nucleoplasmic and soluble than wild-type lamin A/C and accumulates into large intranuclear aggregates in a fraction of cultured patient fibroblasts as well as in cells ectopically expressing either FLAG- or GFP-tagged p.S143P lamin A. In fluorescence loss in photobleaching (FLIP) experiments, non-aggregated EGFP-tagged p.S143P lamin A was significantly more dynamic. In in vitro association studies, p.S143P lamin A failed to form appropriate filament structures but instead assembled into disorganized aggregates similar to those observed in patient cell nuclei. A whole-genome expression analysis revealed an elevated unfolded protein response (UPR) in cells expressing p.S143P lamin A/C. Additional endoplasmic reticulum (ER) stress induced by tunicamycin reduced the viability of cells expressing mutant lamin further. In summary, p.S143P lamin A/C affects normal lamina structure and influences the cellular stress response, homeostasis and viability.

LMNA Mutation c.917T>G (p.L306R) Leads to Deleterious Hyper-Assembly of Lamin A/C and Associates with Severe Right Ventricular Cardiomyopathy and Premature Aging.

Mutations in the LMNA gene coding for the nuclear lamina proteins lamin A and its smaller splice form lamin C associate with a heterogeneous group of diseases collectively called laminopathies. Here, we describe a 2-year-old patient with a previously undescribed phenotype including right ventricular cardiomyopathy, progeroid features, and premature death. Sequencing of LMNA revealed a novel heterozygous de novo mutation p.L306R located in the α-helical rod domain of A-type lamins. Fibroblasts from the patient showed reduced proliferation and early premature replicative senescence, as characterized by progressive hyperlobulation of the nuclei, abnormally clustered centromeres, loss of lamin B1, and reorganization of promyelocytic leukemia nuclear bodies. Furthermore, the patient cells were more sensitive to double-strand DNA breaks. Similar structural and phenotypic defects were observed in normal fibroblasts transfected with FLAG-tagged p.L306R lamin A. Correspondingly, in vitro assembly studies revealed that the p.L306R generates a "hyper-assembly" mutant of lamin A that forms extensive fiber arrays under physiological conditions where wild-type lamin A is still largely soluble. In summary, we report a novel LMNA p.L306R mutation that leads to previously undescribed hyper-assembly of lamin A, heavy distortion of nuclear shape and that manifests as right ventricular cardiomyopathy and premature aging.

Loss of SUFU function in familial multiple meningioma.

Meningiomas are the most common primary tumors of the CNS and account for up to 30% of all CNS tumors. An increased risk of meningiomas has been associated with certain tumor-susceptibility syndromes, especially neurofibromatosis type II, but no gene defects predisposing to isolated familial meningiomas have thus far been identified. Here, we report on a family of five meningioma-affected siblings, four of whom have multiple tumors. No NF2 mutations were identified in the germline or tumors. We combined genome-wide linkage analysis and exome sequencing, and we identified in suppressor of fused homolog (Drosophila), SUFU, a c.367C>T (p.Arg123Cys) mutation segregating with the meningiomas in the family. The variation was not present in healthy controls, and all seven meningiomas analyzed displayed loss of the wild-type allele according to the classic two-hit model for tumor-suppressor genes. In silico modeling predicted the variant to affect the tertiary structure of the protein, and functional analyses showed that the activity of the altered SUFU was significantly reduced and therefore led to dysregulated hedgehog (Hh) signaling. SUFU is a known tumor-suppressor gene previously associated with childhood medulloblastoma predisposition. Our genetic and functional analyses indicate that germline mutations in SUFU also predispose to meningiomas, particularly to multiple meningiomas. It is possible that other genic mutations resulting in aberrant activation of the Hh pathway might underlie meningioma predisposition in families with an unknown etiology.

PP2A methylesterase PME-1 suppresses anoikis and is associated with therapy relapse of PTEN-deficient prostate cancers.

While organ-confined prostate cancer (PCa) is mostly therapeutically manageable, metastatic progression of PCa remains an unmet clinical challenge. Resistance to anoikis, a form of cell death initiated by cell detachment from the surrounding extracellular matrix, is one of the cellular processes critical for PCa progression towards aggressive disease. Therefore, further understanding of anoikis regulation in PCa might provide therapeutic opportunities. Here, we discover that PCa tumours with concomitant inhibition of two tumour suppressor phosphatases, PP2A and PTEN, are particularly aggressive, having < 50% 5-year secondary-therapy-free patient survival. Functionally, overexpression of PME-1, a methylesterase for the catalytic PP2A-C subunit, inhibits anoikis in PTEN-deficient PCa cells. In vivo, PME-1 inhibition increased apoptosis in in ovo PCa tumour xenografts, and attenuated PCa cell survival in zebrafish circulation. Molecularly, PME-1-deficient PC3 cells display increased trimethylation at lysines 9 and 27 of histone H3 (H3K9me3 and H3K27me3), a phenotype known to correlate with increased apoptosis sensitivity. In summary, our results demonstrate that PME-1 supports anoikis resistance in PTEN-deficient PCa cells. Clinically, these results identify PME-1 as a candidate biomarker for a subset of particularly aggressive PTEN-deficient PCa.

A heterozygous p.S143P mutation in LMNA associates with proteasome dysfunction and enhanced autophagy-mediated degradation of mutant lamins A and C.

Lamins A and C are nuclear intermediate filament proteins that form a proteinaceous meshwork called lamina beneath the inner nuclear membrane. Mutations in the LMNA gene encoding lamins A and C cause a heterogenous group of inherited degenerative diseases known as laminopathies. Previous studies have revealed altered cell signaling pathways in lamin-mutant patient cells, but little is known about the fate of mutant lamins A and C within the cells. Here, we analyzed the turnover of lamins A and C in cells derived from a dilated cardiomyopathy patient with a heterozygous p.S143P mutation in LMNA. We found that transcriptional activation and mRNA levels of LMNA are increased in the primary patient fibroblasts, but the protein levels of lamins A and C remain equal in control and patient cells because of a meticulous interplay between autophagy and the ubiquitin-proteasome system (UPS). Both endogenous and ectopic expression of p.S143P lamins A and C cause significantly reduced activity of UPS and an accumulation of K48-ubiquitin chains in the nucleus. Furthermore, K48-ubiquitinated lamins A and C are degraded by compensatory enhanced autophagy, as shown by increased autophagosome formation and binding of lamins A and C to microtubule-associated protein 1A/1B-light chain 3. Finally, chaperone 4-PBA augmented protein degradation by restoring UPS activity as well as autophagy in the patient cells. In summary, our results suggest that the p.S143P-mutant lamins A and C have overloading and deleterious effects on protein degradation machinery and pharmacological interventions with compounds enhancing protein degradation may be beneficial for cell homeostasis.

Divergence of hedgehog signal transduction mechanism between Drosophila and mammals.

The Hedgehog (Hh) signaling pathway has conserved roles in development of species ranging from Drosophila to humans. Responses to Hh are mediated by the transcription factor Cubitus interruptus (Ci; GLIs 1-3 in mammals), and constitutive activation of Hh target gene expression has been linked to several types of human cancer. In Drosophila, the kinesin-like protein Costal2 (Cos2), which associates directly with the Hh receptor component Smoothened (Smo), is essential for suppression of the transcriptional activity of Ci in the absence of ligand. Another protein, Suppressor of Fused (Su(Fu)), exerts a weak negative influence on Ci activity. Based on analysis of functional and sequence conservation of Cos2 orthologs, Su(Fu), Smo, and Ci/GLI proteins, we find here that Drosophila and mammalian Hh signaling mechanisms have diverged, and that, in mouse cells, major Cos2-like activities are absent and the inhibition of the Hh pathway in the absence of ligand critically depends on Su(Fu).

Phosphatase-mediated crosstalk between MAPK signaling pathways in the regulation of cell survival.

Mitogen-activated protein kinase (MAPK) pathways constitute a large modular network that regulates a variety of physiological processes, such as cell growth, differentiation, and apoptotic cell death. The function of the ERK pathway has been depicted as survival-promoting, in essence by opposing the proapoptotic activity of the stress-activated c-Jun NH(2)-terminal kinase (JNK)/p38 MAPK pathways. However, recently published work suggests that extracellular regulated kinase (ERK) pathway activity is suppressed by JNK/p38 kinases during apoptosis induction. In this review, we will summarize the current knowledge about JNK/p38-mediated mechanisms that negatively regulate the ERK pathway. In particular, we will focus on phosphatases (PP2A, MKPs) as inhibitors of ERK pathway activity in regulating apoptosis. A model proposed in this review places the negative regulation of the ERK pathway in a central position for the cellular decision-making process that determines whether cells will live or die in response to apoptosis-promoting signals. In addition, we will discuss the potential functional relevance of negative regulation of ERK pathway activity, for physiological and pathological conditions (e.g., cellular transformation).

p38 Mitogen-activated protein kinase pathway suppresses cell survival by inducing dephosphorylation of mitogen-activated protein/extracellular signal-regulated kinase kinase1,2.

Raf/mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)1,2/extracellular signal-regulated kinase1,2 and MKK3,6/p38 mitogen-activated protein kinase pathways play an important role in cellular survival and apoptosis. The results of this study identify novel mechanisms to explain the opposing effects of these pathways in the regulation of apoptosis induction. Our results show that activation of p38 by adenoviral expression of MKK3b or arsenite treatment was followed by rapid dephosphorylation of MEK1,2 and subsequent apoptosis in human skin fibroblasts. Inhibition of p38 activity by SB203580 and adenoviral expression of dominant-negative forms of p38 potently inhibited MEK1,2 dephosphorylation and apoptosis. Strikingly, p38-mediated dephosphorylation of MEK1,2, was not detected in a series of transformed human cell lines. Taken together, we provide evidence for mechanisms unidentified previously that negatively regulates survival signaling during apoptosis induction. In addition, we show that in all transformed cell lines we have studied thus far, the function of this pathway is impaired.

BARE retrotransposons are translated and replicated via distinct RNA pools.

The replication of Long Terminal Repeat (LTR) retrotransposons, which can constitute over 80% of higher plant genomes, resembles that of retroviruses. A major question for retrotransposons and retroviruses is how the two conflicting roles of their transcripts, in translation and reverse transcription, are balanced. Here, we show that the BARE retrotransposon, despite its organization into just one open reading frame, produces three distinct classes of transcripts. One is capped, polyadenylated, and translated, but cannot be copied into cDNA. The second is not capped or polyadenylated, but is destined for packaging and ultimate reverse transcription. The third class is capped, polyadenylated, and spliced to favor production of a subgenomic RNA encoding only Gag, the protein forming virus-like particles. Moreover, the BARE2 subfamily, which cannot synthesize Gag and is parasitic on BARE1, does not produce the spliced sub-genomic RNA for translation but does make the replication competent transcripts, which are packaged into BARE1 particles. To our knowledge, this is first demonstration of distinct RNA pools for translation and transcription for any retrotransposon.

Activation of p38 alpha MAPK enhances collagenase-1 (matrix metalloproteinase (MMP)-1) and stromelysin-1 (MMP-3) expression by mRNA stabilization.

Here, we have examined the role of distinct MAPK pathways in the regulation of collagenase-1 (matrix metalloproteinase (MMP)-1) and stromelysin-1 (MMP-3) expression by human skin fibroblasts. Tumor necrosis factor-alpha rapidly and transiently activated ERK1/2 and JNK in fibroblasts, whereas the activation of p38 MAPK was more persistent. Inhibition of p38 activity by SB203580 markedly (by 80-90%) inhibited induction of MMP-1 and MMP-3 expression by tumor necrosis factor-alpha, whereas blocking the activation of ERK1/2 by PD98059 had no effect. Activation of endogenous ERK1/2 by adenovirus-mediated transfer of constitutively active MEK1 resulted in potent induction of MMP-1 and MMP-3 expression. Activation of endogenous or adenovirally expressed p38 alpha by adenovirally delivered constitutively active MKK3b and MKK6b also enhanced MMP-1 and MMP-3 expression and augmented the up-regulatory effect of ERK1/2 activation on the expression of these MMPs. Activation of ERK1/2 resulted in induction of c-jun, junB, and c-fos expression, whereas activation of p38 alone had no effect. In contrast, activation of p38 alpha resulted in marked stabilization of MMP-1 and MMP-3 mRNAs. These results identify two distinct and complementary signaling mechanisms mediating induction of MMP-1 and MMP-3 expression in dermal fibroblasts: AP-1-dependent transcriptional activation via the ERK1/2 pathway and AP-1-independent enhancement via p38 alpha MAPK by mRNA stabilization. It is conceivable that both modes of action play an important role in controlling the proteolytic phenotype of fibroblasts, e.g. in wound repair and tumor invasion.