Muscular laminopathies: role of prelamin A in early steps of muscle differentiation.

Lamin A is a nuclear envelope constituent involved in a group of human disorders, collectively referred to as laminopathies, which include Emery-Dreifuss muscular dystrophy. Because increasing evidence suggests a role of lamin A precursor in nuclear functions, we investigated the processing of prelamin A along muscle differentiation. Both protein levels and cellular localization of prelamin A appears to be modulated during C2C12 mouse myoblasts activation. Similar changes also occur in the expression of two lamin A-binding proteins: emerin and LAP2α. Furthermore prelamin A forms a complex with LAP2α in differentiating myoblasts. Prelamin A accumulation in cycling myoblasts by expressing unprocessable mutants affects LAP2α and PCNA amount and increases caveolin 3 mRNA and protein levels, whilst accumulation of prelamin A in differentiated muscle cells following treatment with a farnesyl transferase inhibitor inhibits caveolin 3 expression. These data provide evidence for a critical role of lamin A precursor in the early steps of muscle cell differentiation. In fact the post-translational processing of prelamin A affects caveolin 3 expression and influences the myoblast differentiation process. Thus, altered lamin A processing could affect myoblast differentiation and/or muscle regeneration and might contribute to the myopathic phenotype.

Emerin-prelamin A interplay in human fibroblasts.

BACKGROUND INFORMATION: Emerin is a nuclear envelope protein that contributes to nuclear architecture, chromatin structure, and gene expression through its interaction with various nuclear proteins. In particular, emerin is molecularly connected with the nuclear lamina, a protein meshwork composed of lamins and lamin-binding proteins underlying the inner nuclear membrane. Among nuclear lamina components, lamin A is a major emerin partner. Lamin A, encoded by the LMNA gene (lamin A/C gene), is produced as a precursor protein (prelamin A) that is post-transcriptionally modified at its C-terminal region where the CaaX motif triggers a sequence of modifications, including farnesylation, carboxymethylation, and proteolytic cleavage by ZMPSTE 24 (zinc metalloproteinase Ste24) metalloproteinase. Impairment of the lamin A maturation pathway causing lamin A precursor accumulation is linked to the development of rare diseases such as familial partial lipodystrophy, MADA (mandibuloacral dysplasia), the Werner syndrome, Hutchinson-Gilford progeria syndrome and RD (restrictive dermopathy). RESULTS: In the present study, we show that emerin and different prelamin A forms influence each other's localization. We show that the accumulation of non-farnesylated as well as farnesylated carboxymethylated lamin A precursors in human fibroblasts modifies emerin localization. On the contrary, emerin absence at the inner nuclear membrane leads to unprocessed (non-farnesylated) prelamin A aberrant localization only. Moreover, we observe that the restoration of emerin expression in emerin-null cells induces the recovery of non-farnesylated prelamin A localization. CONCLUSION: These results indicate that emerin-prelamin A interplay influences nuclear organization. This finding may be relevant to the understanding of laminopathies.

Prelamin A is involved in early steps of muscle differentiation.

Lamin A is a nuclear lamina constituent implicated in a number of human disorders including Emery-Dreifuss muscular dystrophy. Since increasing evidence suggests a role of the lamin A precursor in nuclear functions, we investigated the processing of prelamin A during differentiation of C2C12 mouse myoblasts. We show that both protein levels and cellular localization of prelamin A are modulated during myoblast activation. Similar changes of lamin A-binding proteins emerin and LAP2alpha were observed. Furthermore, prelamin A was found in a complex with LAP2alpha in differentiating myoblasts. Prelamin A accumulation in cycling myoblasts by expressing unprocessable mutants affected LAP2alpha and PCNA amount and increased caveolin 3 mRNA and protein levels, while accumulation of prelamin A in differentiated muscle cells following treatment with a farnesyl transferase inhibitor appeared to inhibit caveolin 3 expression. Our data provide evidence for a critical role of the lamin A precursor in the early steps of muscle cell differentiation.

Pre-Lamin A processing is linked to heterochromatin organization.

Pre-lamin A undergoes subsequent steps of post-translational modification at its C-terminus, including farnesylation, methylation, and cleavage by ZMPSTE24 metalloprotease. Here, we show that accumulation of different intermediates of pre-lamin A processing in nuclei, induced by expression of mutated pre-lamin A, differentially affected chromatin organization in human fibroblasts. Unprocessed (non-farnesylated) pre-lamin A accumulated in intranuclear foci, caused the redistribution of LAP2alpha and of the heterochromatin markers HP1alpha and trimethyl-K9-histone 3, and triggered heterochromatin localization in the nuclear interior. In contrast, the farnesylated and carboxymethylated lamin A precursor accumulated at the nuclear periphery and caused loss of heterochromatin markers and Lap2alpha in enlarged nuclei. Interestingly, pre-lamin A bound both HP1alpha and LAP2alpha in vivo, but the farnesylated form showed reduced affinity for HP1alpha. Our data show a link between pre-lamin A processing and heterochromatin remodeling and have major implications for understanding molecular mechanisms of human diseases linked to mutations in lamins.

Effect of Vinorelbine on cell growth and apoptosis induction in human osteosarcoma in vitro.

Vinorelbine (VNR) is a semi-synthetic vinca alkaloid known to exert its antitumour activity by interfering with the polymerisation of tubulin. It has shown a broad spectrum of activity in some advanced carcinomas of lung, breast and ovary. This report demonstrates for the first time the antiproliferative effect of VNR and its molecular mechanism in human osteosarcoma in vitro. TP53 wild-type HOS cells and TP53 mutated MG-63 cells were chosen for this study. In each cell line, VNR caused a significant dose- and time-dependent growth inhibition and induced apoptotic death independent of TP53 status. Phosphorylation and/or alteration of Bcl-2 were not induced by VNR, thereby indicating a new pathway utilised by the drug to induce apoptosis in this tumour in vitro. VNR produced a down-regulation of cyclin D1 and an up-regulation of p53 expression in TP53 wild-type HOS cells, whereas no alteration in cyclin D1 expression was evident in the TP53 negative MG-63 cells. These data suggest a new potential use for Vinorelbine as a therapeutic agent against human osteosarcoma.

Sensitization of multidrug resistant human ostesarcoma cells to Apo2 Ligand/TRAIL-induced apoptosis by inhibition of the Akt/PKB kinase.

Chemotherapeutic agents have been used for the treatment of patients with osteosarcoma (OS). However, inherent or acquired resistance to these agents is a serious problem in the management of OS patients. The emergence of the multidrug resistance (MDR) phenotype in cancer cells is often associated with the overexpression of P-glycoprotein, encoded by the multidrug resistance gene MDR-1. The administration of some of the most common chemotherapeutic agents to these cells becomes ineffective because of their P-gp-driven efflux from the cell. Apo2L/TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines that is considered to induce death of cancer cells but not normal cells. Its powerful apoptotic activity is mediated through its cell surface death domain-containing receptors, TRAIL-R1/DR4 and TRAIL-R2/DR5, which in turn spread the signal in the cytosol through the activation of the caspase cascade. The Akt/PKB kinase is an important cell survival protein which is regulated by D3-phosphoinositides. High Akt expression and activity levels are well documented in many types of tumors, which very often show an altered PI3-K/Akt/PTEN pathway. In this study the U2OS human osteosarcoma cell line and its multidrug resistant (MDR) subline that overexpresses MDR-1 gene, MDR-U2OS, have been analyzed for their responsiveness to TRAIL. In conflict with the presence of active DR4 and DR5 receptors in both clones, U2OS cells exhibited only a low responsiveness to TRAIL, while the MDR-U2OS subline did exhibit a marked TRAIL sensitivity. An analysis of the post-receptor events showed that TRAIL responsiveness correlates with a reduced expression of endogenous Akt. In fact, expression in MDR-U2OS cells of a constitutively active Akt strongly decreased their sensitivity to TRAIL. The identification of Akt as a key modulator of TRAIL responsiveness could help to design TRAIL-based combinations for treatment of osteosarcoma. Moreover, the discovery that multidrug resistant osteosarcomas are highly sensitive to TRAIL-induced apoptosis indicates TRAIL as a new candidate for the treatment of multidrug resistant bone malignancies.