Regulation of nuclear actin levels and MRTF/SRF target gene expression during PC6.3 cell differentiation.

Actin has important functions in both cytoplasm and nucleus of the cell, with active nuclear transport mechanisms maintaining the cellular actin balance. Nuclear actin levels are subject to regulation during many cellular processes from cell differentiation to cancer. Here we show that nuclear actin levels increase upon differentiation of PC6.3 cells towards neuron-like cells. Photobleaching experiments demonstrate that this increase is due to decreased nuclear export of actin during cell differentiation. Increased nuclear actin levels lead to decreased nuclear localization of MRTF-A, a well-established transcription cofactor of SRF. In line with MRTF-A localization, transcriptomics analysis reveals that MRTF/SRF target gene expression is first transiently activated, but then substantially downregulated during PC6.3 cell differentiation. This study therefore describes a novel cellular context, where regulation of nuclear actin is utilized to tune MRTF/SRF target gene expression during cell differentiation.

Regulation of nuclear actin dynamics in development and disease.

Actin has essential functions both in the cytoplasm and in the nucleus, where it has been linked to key nuclear processes, from transcription to DNA damage response. The multifunctional nature of actin suggests that the cell must contain mechanisms to accurately control the cellular actin balance. Indeed, recent results have demonstrated that nuclear actin levels fluctuate to regulate the transcriptional activity of the cell and that controlled nuclear actin polymerization is required for transcription activation, cell cycle progression, and DNA repair. Intriguingly, aberrant nuclear actin regulation has been observed, for example, in cancer, signifying the importance of this process for cellular homeostasis. This review discussed the latest research on how nuclear actin is regulated, and how this influences actin-dependent nuclear processes.

GADD34 mediates cytoprotective autophagy in mutant huntingtin expressing cells via the mTOR pathway.

Increased protein aggregation and altered cell signaling accompany many neurodegenerative diseases including Huntington's disease (HD). Cell stress is counterbalanced by signals mediating cell repair but the identity of these are not fully understood. We show here that the mammalian target of rapamycin (mTOR) pathway is inhibited and cytoprotective autophagy is activated in neuronal PC6.3 cells at 24 h after expression of mutant huntingtin proteins. Tuberous sclerosis complex (TSC) 1/2 interacted with growth arrest and DNA damage protein 34 (GADD34), which caused TSC2 dephosphorylation and induction of autophagy in mutant huntingtin expressing cells. However, GADD34 and autophagy decreased at later time points, after 48 h of transfection with the concomitant increase in mTOR activity. Overexpression of GADD34 counteracted these effects and increased cytoprotective autophagy and cell survival. These results show that GADD34 plays an important role in cell protection in mutant huntingtin expressing cells. Modulation of GADD34 and the TSC pathway may prove useful in counteracting cell degeneration accompanying HD and other neurodegenerative diseases.

Resveratrol reduces oxidative stress and cell death and increases mitochondrial antioxidants and XIAP in PC6.3-cells.

Resveratrol, a polyphenol derived e.g. from red grapes, has been shown to mediate several positive biological actions such as protection of cells against oxidative stress. It can also influence cell signaling, but the mechanisms behind its antioxidant properties are largely unknown. Here we show that RSV reduces oxidative stress and enhances cell survival in PC6.3 cells depending on the concentration. In these cells, RSV increased the levels of antioxidants, SOD2 and TRX2, and of X chromosome-linked inhibitor of apoptosis protein. RSV also activated NFκB signaling as shown using luciferase reporter constructs. These findings show that RSV regulates oxidative stress and mitochondrial antioxidants in neuronal cells. This may contribute to cell protection in various brain disorders.

Downregulation of NF-kappaB signaling by mutant huntingtin proteins induces oxidative stress and cell death.

Accumulation of abnormal proteins and endoplasmic reticulum stress accompany neurodegenerative diseases including Huntington's disease. We show that the expression of mutant huntingtin proteins with extended polyglutamine repeats differentially affected endoplasmic reticulum signaling cascades linked to the inositol-requiring enzyme-1 (IRE1) pathway. Thus, the p38 and c-Jun N-terminal kinase pathways were activated, while the levels of the nuclear factor-kappaB-p65 (NF-kappaB-p65) protein decreased. Downregulation of NF-kappaB signaling was linked to decreased antioxidant levels, increased oxidative stress, and enhanced cell death. Concomitantly, calpain was activated, and treatment with calpain inhibitors restored NF-kappaB-p65 levels and increased cell viability. The calpain regulator, calpastatin, was low in cells expressing mutant huntingtin, and overexpression of calpastatin counteracted the deleterious effects caused by N-terminal mutant huntingtin proteins. These results show that calpastatin and an altered NF-kappaB-p65 signaling are crucial factors involved in oxidative stress and cell death mediated by mutant huntingtin proteins.

Identification of an alternatively spliced isoform of carbonic anhydrase XII in diffusely infiltrating astrocytic gliomas.

Carbonic anhydrase XII (CA XII) is a transmembrane enzyme that is associated with neoplastic growth. CA XII has been proposed to be involved in acidification of the extracellular milieu, creating an appropriate microenvironment for rapid tumor growth. Because RNA sequence databases have indicated that two isoforms of CA XII might exist in human tissues, and because alternatively spliced protein forms have been linked to aggressive behavior of cancer cells, we designed a study to evaluate the presence of the two forms of CA XII in diffuse astrocytomas, a tumor type known for its aggressive and often noncurable behavior. Reverse transcription PCR of tumor samples surprisingly revealed that CA XII present in diffuse astrocytomas is mainly encoded by a shorter mRNA variant. We further showed by Western blotting that anti-CA XII antibody recognized both isoforms in the glioblastoma cell lines, and we then evaluated the expression of CA XII in astrocytomas using immunohistochemistry and correlated the results with various clinicopathological and molecular factors. Of 370 diffusely infiltrating astrocytomas, 363 cases (98%) showed immunoreactions for CA XII. Importantly, CA XII expression correlated with poorer patient prognosis in univariate (p = 0.010, log-rank test) and multivariate survival analyses (p = 0.039, Cox analysis). From these results, we conclude that CA XII is commonly expressed in diffuse astrocytomas and that it might be used as a biomarker of poor prognosis. The absence of 11 amino acids in the shorter isoform, which seems to be common in astrocytomas, may affect the normal quaternary structure and biological function of CA XII.