TGF-β1 modifications in nuclear matrix proteins of osteoblasts during differentiation

Nuclear matrix protein (NMP) composition of osteoblasts shows distinct two-dimensional gel electrophoretic profiles of labeled proteins as a function of stages of cellular differentiation. Because NMPs are involved in the control of gene expression, we examined modifications in the representation of NMPs induced by TGF-β1 treatment of osteoblasts to gain insight into the effects of TGF-β on development of the osteoblast phenotype. Exposure of proliferating fetal rat calvarial derived primary cells in culture to TGF-β1 for 48 h (day 4–6) modifies osteoblast cell morphology and proliferation and blocks subsequent formation of mineralized nodules. Nuclear matrix protein profiles were very similar between control and TGF-β–treated cultures until day 14, but subsequently differences in nuclear matrix proteins were apparent in TGF-β–treated cultures. These findings support the concept that TGF-β1 modifies the final stage of osteoblast mineralization and alters the composition of the osteoblast nuclear matrix as reflected by selective and TGF-β–dependent modifications in the levels of specific nuclear matrix proteins. The specific changes induced by TGF-β in nuclear matrix associated proteins may reflect specialized mechanisms by which TGF-β signalling mediates the alterations in cell organization and nodule formation and/or the consequential block in extracellular mineralization. J. Cell. Biochem. 69:291–303, 1998. © 1998 Wiley-Liss, Inc.     

Chromatin and Cytoskeletal Tethering Determine Nuclear Morphology in Progerin-Expressing Cells

The nuclear morphology of eukaryotic cells is determined by the interplay between the lamina forming the nuclear skeleton, the chromatin inside the nucleus, and the coupling with the cytoskeleton. Nuclear alterations are often associated with pathological conditions as in Hutchinson-Gilford progeria syndrome, in which a mutation in the lamin A gene yields an altered form of the protein, named progerin, and an aberrant nuclear shape. Here, we introduce an inducible cellular model of Hutchinson-Gilford progeria syndrome in HeLa cells in which increased progerin expression leads to alterations in the coupling of the lamin shell with cytoskeletal or chromatin tethers as well as with polycomb group proteins. Furthermore, our experiments show that progerin expression leads to enhanced nuclear shape fluctuations in response to cytoskeletal activity. To interpret the experimental results, we introduce a computational model of the cell nucleus that explicitly includes chromatin fibers, the nuclear shell, and coupling with the cytoskeleton. The model allows us to investigate how the geometrical organization of the chromatin-lamin tether affects nuclear morphology and shape fluctuations. In sum, our findings highlight the crucial role played by lamin-chromatin and lamin-cytoskeletal alterations in determining nuclear shape morphology and in affecting cellular functions and gene regulation.     

Genome-wide nuclear morphology screen identifies novel genes involved in nuclear architecture and gene-silencing in Saccharomyces cerevisiae

Organisation of the cell nucleus is crucial for the regulation of gene expression but little is known about how nuclei are structured. To address this issue, we designed a genomic screen to identify factors involved in nuclear architecture in Saccharomyces cerevisiae. This screen is based on microscopic monitoring of nuclear pore complexes and nucleolar proteins fused with the green fluorescent protein in a collection of approximately 400 individual deletion mutants. Among the 12 genes identified by this screen, most affect both the nuclear envelope and the nucleolar morphology. Corresponding gene products are localised preferentially to the nucleus or close to the nuclear periphery. Interestingly, these nuclear morphology alterations were associated with chromatin-silencing defects. These genes provide a molecular context to explore the functional link between nuclear architecture and gene silencing.     

Nuclear protein modifications in vitellogenic rooster liver

The estrogenic regulation of vitellogenesis in chicken liver provides an unique perspective on cellular reprogramming because males can be induced experimentally. Transient exposure to estradiol (ES) completely alters established patterns of gene expression in rooster hepatocytes within 6 hrs., and triggers major structural and compositional changes in cell nuclei by 24 hrs. Concurrently, the total protein content of nuclei increases nearly 50% and the relative proportion of protein within the nucleoplasmic, chromatin and residual compartments, shifts markedly. These bulk quantitative changes in nuclear composition are accompanied by marked alterations in 2-D electrophoretic patterns of cytoplasmic, nuclear and nuclear matrix polypeptides. Although most individual proteins remain unidentified, several components clearly overlap both the cytoplasmic and nuclear compartments. Reciprocal changes in the 2-D patterns are also evident after ES stimulation, with progressive decline in some and relative increase in other proteins. Among known species, the lamins (La and Lb) decrease in prominence after hormone, while RNP-associated polypeptides become increasingly pronounced in the residual matrix fraction. The results are discussed in relation to other systems where large-scale nuclear reprogramming is known to occur.     

Changes in the Cytoskeletal and Nuclear Matrix Proteins in Rat Hepatocyte Neoplastic Nodules in Their Relation to the Process of Transformation

In a previous paper (Barboroet al.,1993,Biophys. J.65, 1690–1699) we have shown that cancer development in the resistant hepatocyte model of Solt and Farber is characterized by the progressive unfolding of the higher-order structure of chromatin. A possible functional role of decondensation phenomena in cell transformation cannot be ruled out. Genetic activation involves the relaxation of the superstructure of chromatin, which may be, at least in part, modulated by its interaction with the nuclear matrix. Moreover, recent observations suggest that gene expression can be stimulated by alterations in the organization of the cytoskeleton. Therefore, we have characterized the changes in composition that the nuclear matrix–intermediate filament complex undergoes during the evolution of rat hepatocyte nodules. Dramatic changes in the expression of both the nuclear matrix and intermediate filament proteins occur during transformation; they are, however, related in a different way to the stages of carcinogenesis. Several new nuclear matrix proteins appear in early nodules, isolated 9 weeks after initiation. The subsequent evolution of persistent nodules is also characterized by discrete changes in the composition. Thus, the new synthesis of nuclear matrix proteins reflects the emergence of successive cellular populations, in line with the recent finding that a subset of components of the nuclear matrix is cell type-specific. In contrast, intermediate filament proteins undergo continuing changes. A new keratin with apparent molecular weight of 39 kDa, analogous to human keratin 19, appears in early nodules, and its expression steadily increases up to the 32nd week from initiation; at the same time, the amount of the proteolytic fragments of keratins A and D increases sharply. These findings suggest that the inappropriate expression of keratin 19 may be involved in the epigenetic activation of new cellular programs, through the rearrangement of the cytoskeleton which in turn may perturb nuclear matrix function.     

Integrins and stretch activated ion channels; putative components of functional cell surface mechanoreceptors in articular chondrocytes.

Perception of mechanical signals and the biological responses to such stimuli are fundamental properties of load bearing articular cartilage in diarthrodial joints. Chondrocytes utilize mechanical signals to synthesize an extracellular matrix capable of withstanding high loads and shear stresses. Recent studies have shown that chondrocytes undergo changes in shape and volume in a coordinated manner with load induced deformation of the matrix. These matrix changes, together with alterations in hydrostatic pressure, ionic and osmotic composition, interstitial fluid and streaming potentials are, in turn, perceived by chondrocytes. Chondrocyte responses to these stimuli are specific and well coordinated to bring about changes in gene expression, protein synthesis, matrix composition and ultimately biomechanical competence. In this hypothesis paper we propose a chondrocyte mechanoreceptor model incorporating key extracellular matrix macromolecules, integrins, mechanosensitive ion channels, the cytoskeleton and subcellular signal transduction pathways that maintain the chondrocyte phenotype, prevent chondrocyte apoptosis and regulate chondrocyte-specific gene expression.     

Biophysical regulation of histone acetylation in mesenchymal stem cells

Histone deacetylation and acetylation are catalyzed by histone deacetylase (HDAC) and histone acetyltransferase, respectively, which play important roles in the regulation of chromatin remodeling, gene expression, and cell functions. However, whether and how biophysical cues modulate HDAC activity and histone acetylation is not well understood. Here, we tested the hypothesis that microtopographic patterning and mechanical strain on the substrate regulate nuclear shape, HDAC activity, and histone acetylation. Bone marrow mesenchymal stem cells (MSCs) were cultured on elastic membranes patterned with parallel microgrooves 10 μm wide that kept MSCs aligned along the axis of the grooves. Compared with MSCs on an unpatterned substrate, MSCs on microgrooves had elongated nuclear shape, a decrease in HDAC activity, and an increase of histone acetylation. To investigate anisotropic mechanical sensing by MSCs, cells on the elastic micropatterned membranes were subjected to static uniaxial mechanical compression or stretch in the direction parallel or perpendicular to the microgrooves. Among the four types of loads, compression or stretch perpendicular to the microgrooves caused a decrease in HDAC activity, accompanied by the increase in histone acetylation and slight changes of nuclear shape. Knocking down nuclear matrix protein lamin A/C abolished mechanical strain-induced changes in HDAC activity. These results demonstrate that micropattern and mechanical strain on the substrate can modulate nuclear shape, HDAC activity, and histone acetylation in an anisotropic manner and that nuclear matrix mediates mechanotransduction. These findings reveal a new mechanism, to our knowledge, by which extracellular biophysical signals are translated into biochemical signaling events in the nucleus, and they will have significant impact in the area of mechanobiology and mechanotransduction.     

Distinct nuclear gene expression profiles in cells with mtDNA depletion and homoplasmic A3243G mutation

The pathobiochemical pathways determining the wide variability in phenotypic expression of mitochondrial DNA (mtDNA) mutations are not well understood. Most pathogenic mtDNA mutations induce a general defect in mitochondrial respiration and thereby ATP synthesis. Yet phenotypic expression of the different mtDNA mutations shows large variations that are difficult to reconcile with ATP depletion as sole pathogenic factor, implying that additional mechanisms contribute to the phenotype. Here, we use DNA microarrays to identify changes in nuclear gene expression resulting from the presence of the A3243G diabetogenic mutation and from a depletion of mtDNA (rho0 cells). We find that cells respond mildly to these mitochondrial states with both general and specific changes in nuclear gene expression. This observation indicates that cells can sense the status of mtDNA. A number of genes show divergence in expression in rho0 cells compared to cells with the A3243G mutation, such as genes involved in oxidative phosphorylation. As a common response in A3243G and rho0 cells, mRNA levels for extracellular matrix genes are up-regulated, while the mRNA levels of genes involved in ubiquitin-mediated protein degradation and in ribosomal protein synthesis is down-regulated. This reduced expression is reflected at the level of cytosolic protein synthesis in both A3243G and rho0 cells. Our finding that mitochondrial dysfunction caused by different mutations affects nuclear gene expression in partially distinct ways suggests that multiple pathways link mitochondrial function to nuclear gene expression and contribute to the development of the different phenotypes in mitochondrial disease.     

Role of Actin Dependent Nuclear Deformation in Regulating Early Gene Expression

The nucleus of a living cell is constantly undergoing changes in shape and size as a result of various mechanical forces in physiology. These changes correlate with alterations in gene expression, however it is unclear whether nuclear deformation alone is sufficient to elicit these alterations. We used T-cell activation as a model system to test the coupling between nuclear deformation (elongation) and gene expression. Naïve T-cell activation with surrogate antigens resulted in actin dependent nuclear elongation. This was accompanied with Erk and NF-κB signaling to the nucleus to induce CD69 expression. Importantly, inhibiting actin polymerization abolished both nuclear elongation and CD69 expression, while inhibiting Erk, NF-κB or microtubule depolymerization only abolished expression but not elongation. Immobilization of antigen-coated beads, under conditions where actin polymerization was inhibited, rescued both nuclear elongation and CD69 expression. In addition, fibroblast cells plated on fibronectin micropatterns of different sizes showed correlation between nuclear shape index and tenascin C expression. Upon inhibiting the signaling intermediate Erk, tenascin C expression was down regulated although the nuclear shape index remained unaltered. Our results highlight the importance of specific signaling intermediates accompanied with nuclear deformation in the modulation of cellular genomic programs.     

Nuclear dreams: The malignant alteration of nuclear architecture

Cancer is diagnosed by examining the architectural alterations to cells and tissues. Changes in nuclear structure are among the most universal of these and include increases in nuclear size, deformities in nuclear shape, and changes in the internal organization of the nucleus. These may all reflect changes in the nuclear matrix, a non-chromatin nuclear scaffolding determining nuclear form, higher order chromatin folding, and the spatial organization of nucleic acid metabolism. Malignancy-induced changes in this structure may have profound effects on chromatin folding, on the fidelity of genome replication, and on gene expression. Elucidating the mechanisms and the biological consequences of nuclear changes will require the identification of the major structural molecules of the internal nuclear matrix and an understanding of their assembly into structural elements. If biochemical correlates to malignant alterations in nuclear structure can be identified then nuclear matrix proteins and, perhaps nuclear matrix-associated structural RNAs, may be an attractive set of diagnostic markers and therapeutic targets. J. Cell. Biochem. 70:172–180, 1998. © 1998 Wiley-Liss, Inc.     

The genome and the nucleus: a marriage made by evolution

Genomes are housed within cell nuclei as individual chromosome territories. Nuclei contain several architectural structures that interact and influence the genome. In this review, we discuss how the genome may be organised within its nuclear environment with the position of chromosomes inside nuclei being either influenced by gene density or by chromosomes size. We compare interphase genome organisation in diverse species and reveal similarities and differences between evolutionary divergent organisms. Genome organisation is also discussed with relevance to regulation of gene expression, development and differentiation and asks whether large movements of whole chromosomes are really observed during differentiation. Literature and data describing alterations to genome organisation in disease are also discussed. Further, the nuclear structures that are involved in genome function are described, with reference to what happens to the genome when these structures contain protein from mutant genes as in the laminopathies. Review related to the 15th International Chromosome Conference (ICC XV), held in September 2004, Brunel University, London, UK