Expression of the murine alpha B-crystallin gene in lens and skeletal muscle: identification of a muscle-preferred enhancer.

The alpha B-crystallin gene is expressed at high levels in lens and at lower levels in some other tissues, notably skeletal and cardiac muscle, kidney, lung, and brain. A promoter fragment of the murine alpha B-crystallin gene extending from positions -661 to +44 and linked to the bacterial chloramphenicol acetyltransferase (CAT) gene showed preferential expression in lens and skeletal muscle in transgenic mice. Transfection experiments revealed that a region between positions -426 and -257 is absolutely required for expression in C2C12 and G8 myotubes, while sequences downstream from position -115 appear to be determinants for lens expression. In association with a heterologous promoter, a -427 to -259 fragment functions as a strong enhancer in C2C12 myotubes and less efficiently in myoblasts and lens. Gel shift and methylation interference studies demonstrated that nuclear proteins from C2C12 myoblasts and myotubes specifically bind to the enhancer.     

Human bcl-2 gene attenuates the ability of rabbit lens epithelial cells against H2O2-induced apoptosis through down-regulation of the alpha B-crystallin gene

It is well established that the proto-oncogene, bcl-2, can prevent apoptosis induced by a variety of factors. Regarding the mechanism by which BCL-2 prevents cell death, one theory suggests that it acts by protecting cells from oxidative stress. In the lens system, oxidative stress-induced apoptosis is implicated in cataractogenesis. To explore the possibility of anti-apoptotic gene therapy development for cataract prevention and also to further test the anti-oxidative stress theory of BCL-2 action, we have introduced the human bcl-2 gene into an immortalized rabbit lens epithelial cell line, N/N1003A. The stable expression clones of both vector- and bcl-2-transfected cells have been established. Treatment of the two cell lines with H(2)O(2) revealed that bcl-2-transfected cells were less capable of detoxifying H(2)O(2) than the control cells. Moreover, bcl-2-transfected cells are more susceptible to H(2)O(2)-induced apoptosis. To explore why bcl-2-transfected cells have reduced resistance to H(2)O(2)-induced apoptosis, we examined the expression patterns of several relevant genes and found that expression of the alphaB-crystallin gene was distinctly down-regulated in bcl-2-transfected cells compared with that in vector-transfected cells. This down-regulation was specific because a substantial inhibition of BCL-2 expression through antisense bcl-2 RNA significantly restored the level of alphaB-crystallin and, moreover, enhanced the ability of the bcl-2-transfected cells against H(2)O(2)-induced apoptosis. Introduction of a mouse alphaB-crystallin gene into bcl-2-transfected cells also counteracted the BCL-2 effects. Down-regulation of alphaB-crystallin gene was largely derived from changed lens epithelial cell-derived growth factor activity. Besides, alphaB-crystallin prevents apoptosis through interaction with procaspase-3 and partially processed procaspase-3 to prevent caspase-3 activation. Together, our results reveal that BCL-2 can regulate gene expression in rabbit lens epithelial cells. Through down-regulation of the alphaB-crystallin gene, BCL-2 attenuates the ability of rabbit lens epithelial cells against H(2)O(2)-induced apoptosis.     

Human alpha B-crystallin mutation causes oxido-reductive stress and protein aggregation cardiomyopathy in mice

The autosomal dominant mutation in the human alphaB-crystallin gene inducing a R120G amino acid exchange causes a multisystem, protein aggregation disease including cardiomyopathy. The pathogenesis of cardiomyopathy in this mutant (hR120GCryAB) is poorly understood. Here, we show that transgenic mice overexpressing cardiac-specific hR120GCryAB recapitulate the cardiomyopathy in humans and find that the mice are under reductive stress. The myopathic hearts show an increased recycling of oxidized glutathione (GSSG) to reduced glutathione (GSH), which is due to the augmented expression and enzymatic activities of glucose-6-phosphate dehydrogenase (G6PD), glutathione reductase, and glutathione peroxidase. The intercross of hR120GCryAB cardiomyopathic animals with mice with reduced G6PD levels rescues the progeny from cardiac hypertrophy and protein aggregation. These findings demonstrate that dysregulation of G6PD activity is necessary and sufficient for maladaptive reductive stress and suggest a novel therapeutic target for abrogating R120GCryAB cardiomyopathy and heart failure in humans.     

Assignment of the alpha B-crystallin gene to human chromosome 11

Using a human alpha B-crystallin genomic probe and human-mouse somatic cell hybrids, the human alpha B-gene was assigned to chromosome 11 and further corroborated by in situ hybridization to normal metaphase chromosomes. This assignment confirmed and regionally mapped the locus to q22.3-23.1.     

Acid-induced dissociation of alpha A- and alpha B-crystallin homopolymers.

Homopolymers were constructed from the alpha A and alpha B polypeptides isolated from the lens protein alpha-crystallin. As the pH is lowered from 7.0 to 3.4, these homopolymers dissociate to smaller species with molecular masses ranging from 80 to 250 kDa for the alpha A and around 140 kDa for the alpha B dissociation products. The pKa for this dissociation was 3.8 +/- 0.2 for alpha A and 4.1 +/- 0.1 for alpha B homopolymers. Further decreases in pH, to 2.5, resulted in the presence of only denatured alpha B polypeptides, whereas the alpha A dissociation products remained intact. Fractionation of the acid dissociation products from the alpha A homopolymer at pH 2.5 yielded stable species with molecular masses of 220 +/- 30, 160 +/- 20, and 90 +/- 10 kDa. The majority of the population at acid pH consisted of the 160 kDa species. Conformational analysis of these species revealed that most of the secondary structure of the original alpha A homopolymer was retained but that the tertiary structure was perturbed. Fluorescence quenching and energy transfer measurements suggested that the molecule had undergone acid expansion, with the greatest perturbation observed in the smallest particles. The results from this work suggest that alpha A homopolymers are heterogeneous populations of aggregates of a "monomeric" molecule with a molecular mass of 160 kDa. This "monomeric" molecule may be formed from the association of two tetrameric units.     

Chaperone function of mutant versions of alpha A- and alpha B-crystallin prepared to pinpoint chaperone binding sites.

A major stress protein, alpha-crystallin, functions as a chaperone. Site-directed mutagenesis has been used to identify regions of the protein necessary for chaperone function. In this work we have taken some of the previously described mutants produced and assessed their chaperone function by both a traditional heat-induced aggregation method at elevated temperature and using enzyme methods at 37 degrees C. In general the different assays gave parallel results indicating that the same property is being measured. Discrepancies were explicable by the heat lability of some mutants. Most mutants had full chaperone function showing the robust nature of alpha-crystallin. A mutant corresponding to a minor component of rodent alpha A-crystallin, alpha Ains-crystallin, had decreased chaperone function. Decreased chaperone function was also found for human Ser139--> Arg, Thr144-->Arg, Ser59-->Ala mutants of alpha B-crystallin and double mutants Ser45-->Ala/Ser59-->Ala, Lys103--> Leu/His104-->Ile, and Glu110-->His/His111-->Glu. A mutant Phe27-->Arg that was the subject of previous controversy was shown to be fully active at physiological temperatures.     

Cellular distribution of alpha B-crystallin in non-lenticular tissues

alpha B-Crystallin is a subunit of alpha-crystallin, a major protein component of the vertebrate lens. Recently, its expression in various extra-lenticular tissues has been demonstrated by both Western and Northern blotting. In this study, the cellular distribution of alpha B-crystallin in rat organs was examined in detail using immunohistochemistry. Positive reactions were observed in lens, iris, heart, skeletal muscle (type 1 and type 2A fibers), striated muscle in skin and esophagus, Henle's loop and medullary collecting duct of the kidney, Schwann cells of peripheral nerves, glia of the central nervous system, and decidual cells of the placenta. A close correlation with markers of oxidative activity suggests that alpha B-crystallin is expressed in cells that have high levels of oxidative function.     

Differential susceptibility of alpha A- and alpha B-crystallin to gamma-ray irradiation

Alpha-crystallin, a major protein of all vertebrate lenses, consists of two different subunits, alpha A and alpha B, which form polymeric aggregates with an average molecular mass of 300-800 kDa. Both the alpha A and alpha B subunit have a molecular mass of about 20 kDa. It is not known why alpha crystallin aggregates comprise two different subunits, given that the physicochemical properties of these proteins are very similar. The present study compares the susceptibility of the alpha A and alpha B subunits to gamma-rays. We prepared a recombinant form of human alpha A- and alpha B-crystallin and then irradiated the proteins with gamma-rays. Based on far-UV CD spectra, alpha A-crystallin retained beta-sheet conformation after gamma irradiation up to 3.0 kGy, whereas alpha B-crystallin lost beta-sheet conformation upon exposure to gamma irradiation at >1.0 kGy. Size exclusion chromatography showed that the aggregation and polydispersity of recombinant alpha A-crystallin increased slightly after >1.0 kGy irradiation. In contrast, irradiation of alpha B-crystallin at 1.0 kGy resulted in the formation of huge aggregates and a marked increase in heterogeneity. We have also compared the chaperone activities of gamma-irradiated alpha A- and alpha B-crystallin aggregates. The activity of irradiated alpha A-crystallin was retained while that of the irradiated alpha B-crystallin was became inactive after irradiation of >0.5 kGy. Our results indicate that alpha A-crystallin is more stable to gamma irradiation than alpha B-crystallin.     

The in vivo phosphorylation sites of bovine alpha B-crystallin

Phosphate content determinations established that in alpha B-crystallin two phosphate groups can be present in vivo in bovine lenses. Comparison of tryptic digests of phosphorylated and unphosphorylated alpha B chains, revealed the location of the two phosphorylation sites in tryptic peptides T2 and T3. Thermolytic digestion and gas-phase sequencing demonstrated that Ser-19 and Ser-45 are the in vivo phosphorylation sites of bovine alpha B-crystallin. This pattern of phosphorylation differs from the previously reported in vitro obtained results.     

Regulation of the murine alpha B-crystallin/small heat shock protein gene in cardiac muscle.

The murine alpha B-crystallin/small heat shock protein gene is expressed at high levels in the lens and at lower levels in the heart, skeletal muscle, and numerous other tissues. Previously we have found a skeletal-muscle-preferred enhancer at positions -427 to -259 of the alpha B-crystallin gene containing at least four cis-acting regulatory elements (alpha BE-1, alpha BE-2, alpha BE-3, and MRF, which has an E box). Here we show that in transgenic mice, the alpha B-crystallin enhancer directs the chloramphenicol acetyltransferase reporter gene driven by the alpha B-crystallin promoter specifically to myocardiocytes of the heart. The alpha B-crystallin enhancer was active in conjugation with the herpes simplex virus thymidine kinase promoter/human growth hormone reporter gene in transfected rat myocardiocytes. DNase I footprinting and site-specific mutagenesis experiments showed that alpha BE-1, alpha BE-2, alpha BE-3, MRF, and a novel, heart-specific element called alpha BE-4 are required for alpha B-crystallin enhancer activity in transfected myocardiocytes. By contrast, alpha BE-4 is not utilized for enhancer activity in transfected lens or skeletal muscle cell lines. Alpha BE-4 contains an overlapping heat shock sequence and a reverse CArG box [5'-GG(A/T)6CC-3']. Electrophoretic mobility shift assays with an antibody to serum response factor and a CArG-box-competing sequence from the c-fos promoter indicated that a cardiac-specific protein with DNA-binding and antigenic similarities to serum response factor binds to alpha BE-4 via the reverse CArG box; electrophoretic mobility shift assays and antibody experiments with anti-USF antiserum and heart nuclear extract also raised the possibility that the MRF E box utilizes USF or an antigenically related protein. We conclude that the activity of the alpha B-crystallin enhancer in the heart utilizes a reverse CArG box and an E-box-dependent pathway.     

Maintenance of the synthesis of alpha B-crystallin and progressive expression of beta Bp-crystallin in human fetal lens epithelial cells in culture

We have cultured and maintained human fetal lens epithelial cells for several months in primary, secondary, and tertiary culture(s). These cells show unabated synthesis of alpha B-crystallin (alpha B), a lens epithelial cell-specific marker, and progressive expression of beta Bp-crystallin (beta Bp), a major polypeptide of the differentiated lens fiber cells in vivo. Interestingly, the expression of beta Bp was found to be dependent on subculturing of the cells and not on the age of cultures. These observations demonstrate that human fetal lens epithelial cells can be cultured in vitro without the loss of lens specific characteristics and with commitment to differentiation at the biochemical level.     

Identification of a lens-specific regulatory region (LSR) of the murine alpha B-crystallin gene.

Previous studies have shown that the -661/+44 sequence of the murine alpha B-crystallin gene contains a muscle-preferred enhancer (-426/-257) and can drive the bacterial chloramphenicol acetyltransferase (CAT) gene in the lens, skeletal muscle and heart of transgenic mice. Here we show that transgenic mice carrying a truncated -164/+44 fragment of the alpha B-crystallin gene fused to the CAT gene expressed exclusively in the lens; by contrast mice carrying a -426/+44 fragment of the alpha B gene fused to CAT expressed highly in the lens, skeletal muscle and heart, and slightly in the lung, brain, kidney, spleen and liver. DNase I protection experiments indicated that the -147/-118 sequence is protected by nuclear proteins from alpha TN4-1 lens cell line, but not by nuclear proteins from myotubes of the C2C12 cell line. Site directed mutagenesis of this sequence decreased promoter activity in transiently-transfected lens cells, consistent with this sequence being a lens-specific regulatory region (LSR). We conclude that the -426/-257 enhancer is required for expression in skeletal muscle, heart and possibly other tissues, and that the -164/+44 sequence of the alpha B-crystallin gene is sufficient for expression in the lens of transgenic mice.