Multiple antibodies to titin immunoreact with AHNAK and localize to the mitotic spindle machinery.

Recently, the large filamentous striated-muscle protein titin has been observed in non-muscle cells, and, in one instance, has been proposed to have a nuclear function as a chromosomal component contributing to structure and elasticity. In this study, we sought to further characterize the presumptive nuclear isoform of titin. Immunofluorescence microscopy with multiple titin-specific monoclonal antibodies shows localization to the nucleus in interphase cells and to the spindle machinery in mitotic cells in all cell types examined; localization to condensed chromosomes is not observed. An abundant 700-kDa phosphoprotein is the predominant species immunoprecipitated with these antibodies. Sequencing of peptide fragments of the immunopurified protein reveals identity to AHNAK, a nuclear phosphoprotein, an identification that was confirmed by Western blot analysis with antibodies to AHNAK and peptide fragmentation patterns. Sequence comparison suggests similarities between the repetitive heptad phi+/-phiP+/-phi+/- motif in AHNAK and the PEVK region of titin, potentially explaining the cross-reactivity observed between AHNAK antibodies and titin antibodies. Interestingly, although some AHNAK antibodies stain interphase nuclei, no evidence of mitotic spindle localization is seen, suggesting that the identity of the protein at the latter location is more closely related to titin than AHNAK. This concept is further supported by observations that cell lines not expressing AHNAK have similar antititin antibody localization to the mitotic spindle. We conclude that (1) multiple titin antibodies, particularly those recognizing the PEVK region, cross-react with AHNAK, and (2) the mitotic spindle staining observed with antititin antibodies is most likely due to the association of titin or a titin-like molecule with this structure.

Expression of calbindin-D28k in C6 glial cells stabilizes intracellular calcium levels and protects against apoptosis induced by calcium ionophore and amyloid beta-peptide.

The calcium binding protein, calbindin-D28k is normally present in neurons. Recently we reported that brain injury and tumor necrosis factors (TNFs) induce calbindin-D28k in astrocytes. TNF-treated calbindin expressing astrocytes were resistant to acidosis and calcium ionophore toxicity, suggesting that calbindin may have a cytoprotective role in astrocytes in the injured brain (M.P. Mattson, B. Cheng, S.A. Baldwin, V.L. Smith-Swintosky, J. Keller, J. Geddes, Scheff, J.W., Christakos, S., Brain injury and tumor necrosis factors induce calbindin-D28k in astrocytes: evidence for a cytoprotective response, J. Neurosci. Res., 42 (1995) 257). In order to obtain direct evidence for a role of calbindin, using the eukaryotic expression vector pREP4, rat calbindin-D28k was stably expressed in C6 rat astocytoma glial cells. Cytotoxicity in response to calcium ionophore or amyloid beta-peptide (which accumulates in the brain in Alzheimer's disease and has been reported to be neurotoxic) was measured by MTT reduction in vector transfected cells and in calbindin transfected clones. Stably expressed calbindin resulted in increased cell survival in the presence of calcium ionophore (1-10 microM) or amyloid beta-peptide (10-100 microM). In addition, the calcium ionophore or amyloid beta-peptide mediated rise in intracellular calcium in vector transfected cells was significantly attenuated in calbindin expressing cells. Apoptotic cell death was detected by the Hoechst method in vector transfected C6 glial cells treated with calcium ionophore or beta-amyloid (34-36% apoptotic cells/culture). The number of apoptotic nuclei was significantly attenuated in similarly treated calbindin-D28k transfected clones (10-13% apoptotic cells/culture; p<0.01). Our results support the involvement of calcium fluxes in apoptosis and suggest that calbindin-D28k, by buffering calcium, can suppress death in apoptosis susceptible cells in the central nervous system.

Genomic mechanisms involved in the pleiotropic actions of 1,25-dihydroxyvitamin D3.

The biologically active metabolite of vitamin D (cholecalciferol), i.e. 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], is a secosteroid hormone whose mode of action involves stereospecific interaction with an intracellular receptor protein (vitamin D receptor; VDR). 1,25(OH)2D3 is known to be a principal regulator of calcium homeostasis, and it has numerous other physiological functions including inhibition of proliferation of cancer cells, effects on hormone secretion and suppression of T-cell proliferation and cytokine production. Although the exact mechanisms involved in mediating many of the different effects of 1,25(OH)2D3 are not completely defined, genomic actions involving the VDR are clearly of major importance. Similar to other steroid receptors, the VDR is phosphorylated; however, the exact functional role of the phosphorylation of the VDR remains to be determined. The VDR has been reported to be regulated by 1,25(OH)2D3 and also by activation of protein kinases A and C, suggesting co-operativity between signal transduction pathways and 1,25(OH)2D3 action. The VDR binds to vitamin D-responsive elements (VDREs) in the 5' flanking region of target genes. It has been suggested that VDR homodimerization can occur upon binding to certain VDREs but that the VDR/retinoid X receptor (RXR) heterodimer is the functional transactivating species. Other factors reported to be involved in VDR-mediated transcription include chicken ovalbumin upstream promoter (COUP) transcription factor, which is involved in active silencing of transcription, and transcription factor IIB, which has been suggested to play a major role following VDR/RXR heterodimerization. Newly identified vitamin D-dependent target genes include those for Ca2+/Mg(2+)-ATPase in the intestine and p21 in the myelomonocytic U937 cell line. Elucidation of the mechanisms involved in the multiple actions of 1,25(OH)2D3 will be an active area of future research.