Reduced Expression of Lamin A/C Results in Modified Cell Signaling and Metabolism Coupled with Changes in Expression of Structural Proteins

Abstract

Nuclear lamins are intermediate filament proteins that define the shape and stability of nuclei in mammalian cells. In addition to this dominant structural role, recent studies have suggested that the lamin proteins also regulate fundamental aspects of nuclear function. In order to understand different roles played by lamin proteins, we used RNA interference to generate a series of HeLa cell lines to study loss-of-function phenotypes associated with depletion of lamin protein expression. In this study, we used genome-wide proteomic approaches to monitor global changes in protein expression in cells with <10% of normal lamin A/C expression. Of approximately 2000 protein spots analyzed by two-dimensional electrophoresis, only 38 showed significantly altered expression in lamin A/C depleted cells. Of these, 4 protein spots were up-regulated, and 34 were down-regulated. Significant changes were seen to involve the general reduction in expression of cytoskeletal proteins, consistent with altered functionality of the structural cellular networks. At the same time, alterations in expression of proteins involved in cellular metabolism correlated with altered patterns of metabolic activity. In order to link these two features, we used antibody microarrays to perform a focused analysis of expression of cell cycle regulatory proteins. This confirmed a general reduction in expression of proteins regulating cell cycle progression and alteration in signaling pathways that regulate the metabolic activity of cells. The cross-talk between signal transduction and the cytoskeleton emphasizes how structural and kinase-based networks are integrated in mammalian cells to fine-tune metabolic responses.

Identification of Markers for Cellular Stress and Senescence in Laminopathic Cells by Proteomic Analysis

“…Previously reported 2-DE protein profiles of cells depleted of lamin A or expressing laminopathic mutations have revealed 10-50 differentially expressed proteins with identifiable IDs by mass spectral analysis (Chen et al, 2009;Foster et al, 2011;Magagnotti et al, 2012). In our 2-DE analysis of cells expressing GFP-tagged wild-type lamin A or mutant Q294P, 27 proteins could be identified that showed differences in expression.…”

“…The lamina is linked to the cytoskeleton via interactions with nuclear membrane proteins termed nesprins and SUN (Sad1/ UNC-84 homology) domain proteins and disruption of this linkage can cause defects in nuclear positioning and migration during development (Starr and Han, 2003). Changes in abundance of cytoskeletal proteins have been reported in lamindepleted cells and laminopathic cells (Chen et al, 2009;Foster et al, 2011;Magagnotti et al, 2012). Changes in cytoskeletal organization are likely to affect proteins involved in intracellular transport and signalling, like zymogen granule protein 16B and guanine nucleotide binding protein subunit β2 which were reduced in the mutant cells.…”

“…Lamin mutations have been proposed to alter nuclear mechanics, impair gene regulation and cause enhanced proteosomal degradation (Parnaik et al, 2011;Butin-Israeli et al, 2012). Two-dimensional protein profiles of cells with reduced lamin levels or cells from patients with neuromuscular laminopathies have revealed alterations in heat shock proteins, hnRNPs, metabolic pathways and cytoskeletal organization (Chen et al, 2009;Foster et al, 2011;Magagnotti et al, 2012).…”

“…In HeLa cells depleted of lamin A protein by RNA interference, hnRNP proteins like hnRNP-K and hnRNP-C1/C2 and the cytoskeletal proteins keratin and actin were also downregulated (Chen et al, 2009), whereas the protein profile of a colorectal cell line (SW480) depleted of lamin A showed decrease in nucleophosmin, HSPD1 and HSPA9, and upregulation of β-actin and vimentin (Foster et al, 2011). In a proteomic study with skin fibroblasts from patients with neuromuscular laminopathies, proteins downregulated in the patients cells which were also reduced in our lamin mutant Q294P samples included α-enolase, HSP90, hnRNP C1/C2, L-lactate dehydogenase B chain, vimentin and β-tubulin (Magagnotti et al, 2012).…”

Label-free mass spectrometry exploits dozens of detected peptides to quantify lamins in wildtype and knockdown cells

“…In agreement with recent findings, the perturbation to the A549 proteome is relatively slight in response to a lamin KD to ~30% of WT. 47 The perturbation is broader in MSCs in response to a KD of similar magnitude. A breakdown of detectable proteins by function indicates that perturbation to the proteome in MSCs is primarily focused in two areas: upregulation of nuclear proteins (typically proteins involved with mRNA splicing and processing, chromatin binding proteins, and helicases) and downregulation of cytoskeletal and structural proteins (typically actin binding proteins).…”