Expression of small heat shock protein alpha B-crystallin is induced after hepatic stellate cell activation

Alon Lang1, Laura W. Schrum2, Robert Schoonhoven3, Shmuel Tuvia4, Jose A. Solís-Herruzo1, Hidekazu Tsukamoto5, David A. Brenner1,6, and Richard A. Rippe1

Departments of 1 Medicine, 2 Surgery, 3 Environmental Science and Engineering, and 6 Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599; and 4 Department of Cell Biology, Duke University, Durham, North Carolina 27710; and 5 Departments of Medicine and Pathology, University of Southern California, Los Angeles, California 90033


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Using the differential PCR display method to select cDNA fragments that are differentially expressed after hepatic stellate cell (HSC) activation, we have isolated from activated HSCs a cDNA that corresponds to rat alpha B-crystallin. Northern blots confirmed expression of alpha B-crystallin in culture-activated HSCs but not in quiescent HSCs. Western blot analysis and immunocytochemical staining confirmed expression of alpha B-crystallin protein in activated but not quiescent HSCs. alpha B-crystallin is induced as early as 6 h after plating HSCs on plastic and continues to be expressed for 14 days in culture. Expression of alpha B-crystallin was also induced in vivo in activated HSCs from experimental cholestatic liver fibrosis. Confocal microscopy demonstrated a cytoplasmic distribution of alpha B-crystallin in a cytoskeletal pattern. Heat shock treatment resulted in an immediate perinuclear redistribution that in time returned to a normal cytoskeletal distribution. The expression pattern of alpha B-crystallin was similar to that of HSP25, another small heat shock protein, but differed from the classic heat shock protein HSP70. Therefore, alpha B-crystallin represents an early marker for HSC activation.

differential polymerase chain reaction display; gene expression; liver fibrosis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN LIVER FIBROSIS, THE SYNTHESIS and deposition of several extracellular matrix proteins are increased. Increased extracellular matrix production alters the normal architecture of the liver and inhibits its functions. The perisinusoidal hepatic stellate cell (HSC) is largely responsible for this increase in extracellular matrix deposition. The HSC after a fibrogenic stimulus is transformed from a quiescent vitamin A-storing cell type to that of an activated cell type (see Ref. 11 for review). Accompanying HSC activation are numerous changes in cellular morphology and in the pattern of gene expression. These changes include a loss of the stored retinoids, an increase in rough endoplasmic reticulum, enhanced cell proliferation, and dramatic increases in synthesis and deposition of several extracellular matrix proteins, of which type I collagen is predominant. HSC activation is observed in several in vivo models of experimentally induced liver fibrosis, including alcohol-induced liver fibrosis (52) and cholestatic liver injury induced by ligation of the common bile duct (43). Several key features of HSC activation can be recapitulated by culturing the cells on plastic, and this offers a convenient model system to study HSC activation.

Differential PCR display (DD-PCR) (33) provides a convenient and sensitive method to assess differential gene expression after HSC activation. This method allows for the detection of mRNAs that are either increased or decreased after cellular changes. Using this technique, we (14) have previously demonstrated that the expression of intercellular adhesion molecule-1 is induced after activation of the HSC, both in vitro and in vivo. Using DD-PCR as the initial screening method, we now report that expression of alpha B-crystallin is induced after both in vitro and in vivo activation of HSCs.

alpha B-crystallin is a major component of the vertebrate eye lens but is also present at lower levels in other tissues, such as the heart, skeletal muscle, kidney, placenta, skin, brain, peripheral nerves, spinal cord, retina, and liver (8, 16, 20). alpha B-crystallin belongs to the family of small heat shock proteins and displays a molecular chaperone activity under stress conditions (8, 24, 32). The expression of alpha B-crystallin in the HSC was similar to that of HSP25, another small heat shock protein. Expression of both proteins was induced after HSC activation, but heat shock did not result in an appreciable increase in protein levels. On the other hand, expression of HSP70, the classic heat shock protein, was not induced after HSC activation, whereas heat shock resulted in persisted induction of HSP70 protein and transient induction of HSP70 mRNA in cultured HSCs. Therefore, expression of alpha B-crystallin is induced early and persistently after HSC activation.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HSC isolation and culture. HSCs were isolated from adult male Sprague-Dawley rats (>400 g) using sequential pronase-collagenase digestion followed by arabinogalactan (Larcoll, Sigma Chemical, St. Louis, MO) density gradient centrifugation as previously described (48). For isolation of HSCs from cholestatic rats, a mixture of 0.02% type IV collagenase and 0.04% type I collagenase (Boehringer Mannheim, Indianapolis, IN) was used during the perfusion. Isolated HSCs were cultured in DMEM (GIBCO BRL, Gaithersburg, MD) supplemented with 10% FCS in a 5% CO2-95% air atmosphere. All animal procedures were performed under the guidelines set by the University of North Carolina Institutional Animal Care and Use Committee and the Institutional Care and Use Committee of the University of Southern California and are in accordance with the Guide for the Care and Use of Laboratory Animals [DHEW Publication No. (NIH) 85-23, revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20505].

Bile duct ligation. Male Wistar rats (body wt, 550-600 g) underwent common bile duct ligation and transection as previously described (14). To serve as controls, another group of animals underwent sham operations.

DD-PCR analysis. Total RNA was obtained from freshly isolated quiescent HSCs and from culture-activated HSCs using acidified phenol as previously described (48). The DD-PCR method was performed essentially as described by Liang and Pardee (33). Briefly, first-strand cDNA was generated in a reaction containing 2 µg of total RNA, 20 µmol/l dNTPs, 1 µmol/l of a poly-T primer (5'-TTTTTTTTTTTGC-3'), 50 mmol/l Tris · HCl, pH 8.3, 75 mmol/l KCl, 3 mmol/l MgCl2, and 10 mmol/l dithiothreitol (DTT). The reaction mixture was heated at 65°C for 5 min and then at 37°C for 10 min. Murine leukemia virus RT (200 U; GIBCO BRL) was added, and the reaction was incubated at 37°C for an additional 50 min. One-tenth of the first-strand cDNA synthesis reaction was subjected to PCR amplification using the poly-T primer described above along with a 10-nucleotide primer (5'-GTTCCATACG-3') in a reaction mixture containing 2 µmol/l dNTPs, 1 µmol/l of each primer, 10 mmol/l KCl, 0.01 mCi [alpha -35S]-dATP (1,000 Ci/mmol; ICN Biomedicals, Costa Mesa, CA), and 2.5 U of Taq DNA polymerase (Boehringer Mannheim). The PCR reaction was performed with 40 cycles at 94°C for 30 s, 40°C for 2 min, and 72°C for 30 s followed by an extension incubation at 72°C for 10 min followed by a 4°C incubation using a GeneAmp PCR system 9600 (Perkin-Elmer Cetus, Emeryville, CA). An aliquot of the PCR reaction was run in a 4% sequencing gel and dried without fixing. After autoradiography at room temperature for 24 h, the autoradiogram was carefully aligned with the dried gel, and differentially expressed bands were excised. The DNA was eluted from the gel by incubating the gel slice in sterile, distilled water for 10 min, followed by boiling for 15 min. Afterwards, the DNA was precipitated by adding 100 µg/ml glycogen, 0.1 vol of 3 mol/l sodium acetate, and 2.5 vol of 100% ethanol and incubating at -20°C for 1 h. The sample was centrifuged at room temperature for 10 min, and the pellet was washed with 75% ethanol and dried. The eluted DNA fragment was suspended in sterile distilled water, and an aliquot was reamplified using the same primers, PCR conditions, and amplification parameters as described above. PCR amplification products were visualized by electrophoresing an aliquot of the PCR reaction in a 5% nondenaturing polyacrylamide gel, and DNA bands were visualized by ethidium bromide staining. The PCR products were cloned into the TA cloning vector (Invitrogen, San Diego, CA) following the manufacturer's recommended protocol. Cloned PCR fragments were sequenced using the Sequenase DNA sequencing kit version 2 (United States Biochemical, Cleveland, OH). A computer search was performed with the nucleotide sequence of each clone using the National Center for Biotechnology Information BLAST network service.

Northern blot analysis. Northern blot analysis was performed using total RNA as previously described (48). Random primer radiolabeled probes were generated from gel-purified, cloned PCR cDNA fragments of alpha B-crystallin and a cDNA insert of human HSP70 (13) using a random primer radiolabeling kit (RediPrime kit, Boehringer Mannheim).

Western blot analysis. Whole cell extracts were prepared using Dignam C buffer (49). Protein samples (20 µg/lane) were electrophoresed in a 10% acrylamide gel and electrophoretically transferred to a nitrocellulose membrane. The membrane was blocked using 5% nonfat milk in TBS-T (25 mmol/l Tris · HCl, pH 8.0, 144 mmol/l NaCl, and 0.75% Tween 20) at room temperature for 30 min then incubated with either alpha B-crystallin, HSP25, or HSP70 antibodies (1:1,000) (StressGen Biotechnology, Victoria, BC, Canada), each diluted in 5% nonfat milk in TBS-T at room temperature for 2 h. Afterwards, the membrane was washed two times in TBS-T for 5 min. Alkaline phosphatase-conjugated anti-rabbit antibody diluted 1:1,000 in 5% nonfat milk in TBS-T (Santa Cruz Biotechnology, Santa Cruz, CA) was incubated with the membrane at room temperature for 2 h and then washed as described above. Signals were detected using 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium as the substrate. In the Western blot confirming that the phosphoserine antibody recognizes rat phosphoserine proteins, 50 µg of whole cell extract were obtained from Rat-1 cells, and the proteins were electrophoretically separated in a 10% SDS-polyacrylamide gel. The proteins were transferred to a polyvinylidine difluoride membrane (Immobilon-P, Millipore, Bedford, MA). The membrane was blocked at room temperature for 1 h using 3% BSA in TBS-T (10 mmol/l Tris · HCl, pH 7.5, 50 mmol/l NaCl, and 0.1% Tween 20). The membrane was probed with phosphoserine antibody (1:500 in 3% BSA in TBS-T) at room temperature for 1 h and washed in TBS-T twice at room temperature for 20 min. The secondary antibody, anti-rabbit horseradish peroxidase (HRP) (1:1,000 in 3% BSA in TBS-T) was incubated with the membrane at room temperature for 30 min and washed as described above. Signals were detected using the enhanced chemiluminescence (ECL) detection kit (Amersham Life Sciences, Arlington Heights, IL).

Immunoprecipitations. The immunoprecipitation assays were performed as previously described (49). Briefly, whole cell extracts were made from culture-activated HSCs by lysing cell pellets in RIPA buffer (20 mmol/l Tris · HCl, pH 7.5, 150 mmol/l NaCl, 2 mmol/l EDTA, 1% sodium deoxycholate, 1% Triton X-100, and 0.25% SDS). Extracts were precleared using normal rabbit serum overnight at 4°C. Immune complexes were collected by adding protein A agarose and briefly centrifuged at room temperature. The supernatant was collected, phosphoserine antibody (5 µg; Zymed Laboratories, South San Francisco, CA) or alpha B-crystallin antibody (2 µl; StressGen Biotechnology) was added, and the reaction mixture was incubated at 4°C overnight. Immune complexes were collected, as described above, boiled for 5 min in 2× SDS buffer (100 mmol/l Tris · HCl, pH 6.8, 200 mmol/l DTT, 4% SDS, 0.2% bromophenol blue, and 20% glycerol), and electrophoresed in a 10% SDS-polyacrylamide gel. In addition, cellular extracts (10 µg/lane) obtained from quiescent and culture-activated HSCs were electrophoresed in the gel. Samples were electrophoretically transferred to a nitrocellulose membrane, and the membrane was blocked at room temperature for 30 min using 5% nonfat milk in TBS-T (25 mmol/l Tris · HCl, pH 8.0, 144 mmol/l NaCl, and 0.75% Tween 20). The membrane was probed with alpha B-crystallin antibody (1:1,000; StressGen Biotechnology) at room temperature for 2 h and then washed three times in TBS-T. The membrane was incubated in anti-rabbit HRP (1:1,000, Santa Cruz Biotechnology) at room temperature for 30 min and washed as described above. Signals were detected using the ECL detection kit (Amersham Life Sciences).

Immunocytochemistry. Freshly isolated HSCs were seeded (5 × 104 cells/well) in six-well dishes that contained coverslips. After 14 days in culture, the cells were fixed in methanol and washed three times with wash solution (PBS containing 1% goat serum, 1% BSA, and 0.2% Tween 20). The cells were then incubated with wash solution containing 0.5% Triton X-100 at room temperature for 10 min to permeabilize the cells. Afterward, the cells were washed three times and then incubated at room temperature for 3 h with the primary antibody diluted in wash solution. Primary antibodies and dilutions used were alpha B-crystallin, 1:1,000 (StressGen Biotechnologies); smooth muscle alpha -actin, 1:100 (DAKO, Capinteria, CA); vimentin, 1:100 (Sigma Chemical); paxillin, 1:100 (Santa Cruz Biotechnology); alpha -tubulin, 1:100 (Santa Cruz Biotechnology); glial fibrillar acidic protein (GFAP), 1:250 (BioGenex, San Ramon, CA); and foiden, 1:100 (gift from Dr. Keith Burridge, University of North Carolina, Chapel Hill). The cells were washed three times and then incubated with FITC-conjugated anti-rabbit antibody or rhodamine-conjugated anti-mouse antibody (each diluted 1:200 in wash solution) at room temperature for 1 h. The cells were washed three times, and the coverslips were mounted on glass slides using Vectashield mounting medium (Vector Laboratories, Burlingame, CA). To costain for alpha B-crystallin and F-actin, the cells were incubated with the primary and secondary antibodies to detect for alpha B-crystallin, as described above, then incubated with 165 nmol/l rhodamine phalloidin (Molecular Probes, Eugene, OR) diluted in PBS at room temperature for 20 min. The cells were washed three times and mounted on glass slides as described above. Cells were visualized using confocal microscopy.

RT-PCR. HSCs were isolated from the livers of sham-operated and bile duct-ligated rats, and total RNA was isolated from the cells as described above. First-strand cDNA was generated using 2 µg of RNA in a reaction mixture containing 10 mmol/l oligo(dT)15 primer, 50 mmol/l Tris · HCl, pH 8.3, 1 mmol/l of each dNTP, 75 mmol/l KCl, 3 mmol/l MgCl2, 10 mmol/l DTT, and 25 U RNase inhibitor (Boehringer Mannheim). The reaction mixture was incubated at 68°C for 2 min, then at 4°C for 2 min during which murine leukemia virus RT (600 U; GIBCO BRL) was added. The reaction mixture was incubated at 42°C for 60 min. The synthesized cDNA was used in a PCR reaction using specific sets of primers for alpha B-crystallin or beta -actin, as an internal control, based on previously published sequences for alpha B-crystallin (Ref. 3; GenBank accession no. X60352) and beta -actin (54). The nucleotide sequences for the primers used for alpha B-crystallin amplification were as follows: sense strand, 5'-CTGACCTCTTCTCTACAGCCACT-3' (nt 101-123) and antisense strand, 5'-CGTGCACCTCAATCACGTCTCC-3' (nt 301-280). The nucleotide sequence for the beta -actin primers were as follows: sense strand, 5'-GAGCTATGAGCTGCCTGACG-3' (nt 801-820) and antisense strand, 5'-AGCACTTGCGGTCCACGATG-3' (nt 1045-1026). Each PCR reaction contained 1 µmol/l of each specific primer pair, 10 mmol/l Tris · HCl, pH 8.3, 200 µmol/l of each dNTP, 2 mmol/l MgCl2, 50 mmol/l KCl, and 2.5 U Taq DNA polymerase. The PCR reaction was cycled as follows: an initial denaturation of the cDNA sample at 94°C for 3 min, followed by 23 cycles at 94°C for 45 s, 55°C for 30 s, 72°C for 90 s, and a final extension incubation at 72°C for 10 min.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DD-PCR reveals that alpha B-crystallin is differentially expressed after HSC activation. Using the DD-PCR technique, we have isolated a bank of cDNA fragments whose expression is either induced or suppressed after HSC activation in culture. The nucleotide sequence of one of the differentially expressed cDNA fragments that we isolated, ~495 bp in size, was 97% homologous to the 5' and 3' portions of the rat alpha B-crystallin cDNA (GenBank accession no. X60352). This isolated cDNA fragment encompassed nt 46-657 of the reported alpha B-crystallin cDNA sequence. To confirm that alpha B-crystallin is differentially expressed after HSC activation, we performed Northern blot analysis using total RNA obtained from quiescent and culture-activated HSCs. alpha B-crystallin expression was detected in activated HSCs but not in quiescent HSCs (Fig. 1A). The size of the alpha B-crystallin transcript was ~1200 nt, similar to that reported for alpha B-crystallin in other non-lens tissue (18). Western blot analysis was used to demonstrate differential expression of alpha B-crystallin protein after HSC activation. The presence of alpha B-crystallin protein was detected in activated HSCs but not in quiescent HSCs (Fig. 1B).


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 1.   Differential expression of alpha B-crystallin in activated and quiescent hepatic stellate cells (HSCs). A: Northern blot analysis was performed using total RNA (10 µg/lane) from freshly isolated HSCs (day 0; Q) and HSCs cultured on plastic for 15 days (day 15; A). The blot was probed using the alpha B-crystallin cDNA fragment isolated from differential PCR display or an isolated cDNA fragment from the beta -actin gene to serve as a control for RNA loading. B: Western blot analysis was performed using total cellular proteins (10 µg/lane) from freshly isolated HSCs (Q) and HSCs cultured on plastic for 15 days (A). The immunoblot was probed using an alpha B-crystallin antibody. Equal loading of the filter was confirmed by staining the filter with Ponceau S (data not shown).

alpha B-crystallin is expressed early after HSC activation. To assess when alpha B-crystallin is induced during HSC activation, cells were isolated, plated on plastic, and incubated for various time periods. The cells were fixed and stained immunocytochemically for alpha B-crystallin. Within 6 h after plating HSCs, alpha B-crystallin was detected and displayed a focal expression pattern (Fig. 2). Over time, the cellular pattern of expression became increasingly diffused, and after 7 days in culture the localization pattern became cytoskeletal like in appearance. Expression of alpha B-crystallin persisted at high levels throughout 14 days of culture.


View larger version (94K):
[in this window]
[in a new window]
 
Fig. 2.   alpha B-crystallin is expressed within 6 h after HSC isolation. HSCs were isolated and fixed at different time points. Immunofluorescence was performed using anti-alpha B-crystallin antibody followed by a FITC-conjugated detection antibody. Images were captured using confocal microscopy. Magnification, ×400.

Cellular distribution of alpha B-crystallin demonstrates cytoskeletal pattern. alpha B-crystallin has been shown to colocalize with several cytoskeletal proteins in other cell types. These include associations with actin and desmin in cells derived from the lens and heart (2, 6, 34, 56), GFAP, vimentin, and desmin in astrocytoma cells (45), and associations with vimentin and peripherin in cellular extracts (10). We attempted to colocalize alpha B-crystallin with several cytoskeletal proteins in the activated HSC. HSCs were isolated and plated for 14 days. The cells were trypsinized and seeded onto glass coverslips and grown for 2 days, then fixed and costained for alpha B-crystallin and smooth muscle alpha -actin (Fig. 3, A-C), F-actin (Fig. 3, D-F), vimentin (Fig. 3, G-I), paxillin (Fig. 3, J-L), or alpha -tubulin (Fig. 3, M-O). In addition, we performed costaining experiments for alpha B-crystallin and GFAP or foiden (data not shown). Although the pattern of alpha B-crystallin expression is cytoskeletal in nature, we were unable to show colocalization with the different cytoskeletal proteins that we examined.


View larger version (76K):
[in this window]
[in a new window]
 
Fig. 3.   Costaining alpha B-crystallin with other cellular proteins. HSCs were cultured for 15 days on coverslips. Cells were fixed onto the coverslips and immunostained with a rhodamine-conjugated antibody (red) (or using rhodamine phalloidin for F-actin) against smooth muscle alpha -actin (A), F-actin (D), vimentin (G), paxillin (J), or alpha -tubulin (M) or stained with a FITC-conjugated anti-alpha B-crystallin antibody (green) (B, E, H, K, and N). Cells were costained with alpha B-crystallin and smooth muscle alpha -actin (C), F-actin (F), vimentin (I), paxillin (L), or alpha -tubulin (O). No definite colocalization was noted. Images were captured using confocal microscopy. Magnification, ×400.

Cellular distribution of alpha B-crystallin changes after heat shock treatment of HSCs. alpha B-crystallin is a member of the small heat shock protein family (8). These proteins function to protect other proteins in the cell from detrimental effects of excess heat and stress. To assess the cellular location of alpha B-crystallin after heat shock, culture-activated HSCs were subjected to heat shock and then immunocytochemically stained for alpha B-crystallin after different time periods following heat shock. Dramatic changes were noted in the distribution pattern for alpha B-crystallin after heat shock. Within 1 h after heat shock recovery the location of alpha B-crystallin became perinuclear. After 2 h of heat shock recovery, the cellular distribution of alpha B-crystallin became globular in appearance, and within 24 h, the normal cellular distribution pattern for alpha B-crystallin was restored (Fig. 4). This cellular redistribution pattern after heat shock represents a typical response for heat shock proteins (10).


View larger version (129K):
[in this window]
[in a new window]
 
Fig. 4.   The subcellular distribution of alpha B-crystallin changes after heat treatment. Activated HSCs were heat shocked at 42°C for 1 h, and the cells were allowed to recover at 37°C. The cells were fixed and immunohistochemically stained for alpha B-crystallin before heat treatment (0) or 1, 2, or 24 h after heating. The fibrillar expression pattern of alpha B-crystallin transforms to a nodular pattern within 2 h after heating and returns to its normal cellular distribution pattern within 24 h after the heat treatment. Images were captured using confocal microscopy. Magnification, ×400.

alpha B-crystallin is not phosphorylated in activated HSC. It has been shown that alpha B-crystallin becomes phosphorylated after stress conditions (17). Three specific serine residues are targeted for phosphorylation. To assess whether culture-induced activation of HSCs results in serine phosphorylation of alpha B-crystallin, we performed immunoprecipitations using extracts obtained from culture-activated HSCs. Extracts were immunoprecipitated with an alpha B-crystallin antibody or a phosphoserine antibody and the immunoprecipitated proteins probed for alpha B-crystallin. When the alpha B-crystallin antibody was used for immunoprecipitation, alpha B-crystallin was detected in the immunoprecipitated proteins (Fig. 5A). However, when the extracts were immunoprecipitated with the phosphoserine antibody, alpha B-crystallin was not detected in the immunoprecipitated proteins (Fig. 5B). As a control, cellular extracts from quiescent and culture-activated HSCs were included in the Western blot showing expression of alpha B-crystallin in activated HSCs and not in quiescent HSCs. In addition, cellular extracts from Rat-1 cells were probed using the phosphoserine antibody to confirm that the antibody recognized rat phosphoserine proteins (Fig. 5C). This data demonstrates that alpha B-crystallin is not phosphorylated in culture-activated HSCs.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 5.   alpha B-crystallin (alpha B-crys) is not phosphorylated in culture-activated HSCs. A Western blot for alpha B-crystallin was performed using cellular extracts obtained from culture-activated HSCs that were immunoprecipitated (IP) for alpha B-crystallin (A, lane 1) or phosphorylated serine proteins (B, lane 1; alpha -P-ser). As controls in the Western blot, cellular extracts (10 µg/lane) from quiescent HSCs (Q; lane 2 in A and B) and culture-activated HSCs (A; lane 3 in A and B) were included in the Western blot. Both membranes were immunoblotted (IB) for alpha B-crystallin. C: a Western blot was performed using whole cell extracts (WCE; 50 µg/lane) obtained from Rat-1 cells and probed using the phosphoserine antibody to demonstrate that the phosphoserine antibody recognizes rat serine phosphorylated proteins.

Expression of alpha B-crystallin is increased in HSCs isolated after fibrogenic stimulus in vivo. To determine if alpha B-crystallin gene expression is induced after a fibrogenic stimulus in vivo, bile duct ligations in rats were performed. alpha B-crystallin gene expression in HSCs was assessed by RT-PCR. Only low levels of alpha B-crystallin mRNA were detected in HSCs isolated from sham-operated rats, whereas HSCs isolated from bile duct-ligated rats demonstrated significantly higher levels of alpha B-crystallin mRNA (Fig. 6). Together, these data demonstrate that alpha B-crystallin expression is induced in HSCs when activated in vitro by culture and in vivo after a fibrogenic stimulus.


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 6.   alpha B-crystallin is expressed in vivo after bile duct ligation (BDL). Rats were either sham operated or underwent common BDL. Three weeks after BDL, HSCs and total cellular RNA were isolated. RT-PCR analysis was performed using alpha B-crystallin or beta -actin primers. Lanes 1 and 2 are positive and negative controls, respectively. Lanes 3-7 are samples obtained from sham-operated rats, and lanes 8-12 are samples from BDL-operated rats. The RT-PCR reactions were analyzed during the log phase of product amplification. M, molecular mass marker.

Expression of alpha B-crystallin and HSP25 in HSC is similar but significantly differs from that of HSP70. Because alpha B-crystallin belongs to the family of small heat shock proteins, we compared the expression pattern of alpha B-crystallin to HSP25, another small heat shock protein family member, and to HSP70, the classic heat shock protein. Western blot analysis using proteins isolated from quiescent and culture-activated HSCs showed that expression of alpha B-crystallin and HSP25 is induced after HSC activation in culture (Fig. 7). After a 1-h heat treatment of activated HSCs at 42°C followed by recovery at 37°C, protein expression of either alpha B-crystallin or HSP25 was not significantly increased following heat treatment. On the other hand, expression of HSP70 was not induced after HSC activation in culture but was persistently induced for 24 h after the heat shock (Fig. 7).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 7.   The expression pattern of alpha B-crystallin and the heat shock protein HSP25 is similar in HSCs but differs from that of HSP70. Western blot analysis was performed using total cellular proteins (20 µg/lane) obtained from quiescent HSCs (Q), culture-activated HSCs (A), or activated HSCs that were heat treated at 42°C for 1 h and then allowed to recover at 37°C for 3, 6, 12, or 24 h. The filters were immunoblotted using a HSP70, HSP25, or an alpha B-crystallin antibody. Equal loading of the filters was confirmed by staining the filter with Ponceau S (data not shown).

In accordance with the protein results, expression of alpha B-crystallin mRNA remained nearly unchanged after heat shock (Fig. 8). HSP70 mRNA was not detected in activated HSCs; however, transient expression of HSP70 mRNA was observed after heat shock (Fig. 8). Three hours after the heat shock, HSP70 mRNA levels dramatically increased but quickly diminished. Six hours after the heat shock, HSP70 mRNA levels were weakly detectable, and no expression was observed 12 and 24 h after the heat shock. Together, these data demonstrate that differential expression of heat shock proteins occurs in HSCs. The small heat shock proteins alpha B-crystallin and HSP25 are induced after HSC activation and do not show a substantial increase in expression following heat shock. In contrast, the classic heat shock protein HSP70 is not induced after HSC activation but is induced following heat shock treatment of activated HSCs.


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 8.   Expression of alpha B-crystallin and HSP70 mRNA differs after heat treatment of activated HSCs. Northern blot analysis was performed using total RNA (5 µg/lane) isolated from culture-activated HSCs not heat treated (0) or from cells heat treated at 42°C for 1 h and then allowed to recover at 37°C for 3, 6, 12, or 24 h. The blot was probed for HSP70 (A) or for alpha B-crystallin (B). C: ethidium-stained gel demonstrating equal loading of the samples.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To gain a better understanding of the molecular events that take place after HSC activation, we have been using the DD-PCR technique (33) to isolate genes that are either activated or suppressed after HSC activation in culture. In this study, we demonstrated that expression of alpha B-crystallin is induced within 6 h after plating the cells and thus represents one of the earliest induced genes identified after HSC activation. The transcription factor KLF6, previously referred to as Zf9, has also been shown to be an early induced gene after HSC activation in vivo (47). We also demonstrated that alpha B-crystallin gene expression is induced in HSCs that are activated in vivo after cholestatic liver injury induced by common bile duct ligation. After 1 h of heat treatment, the cellular distribution of alpha B-crystallin changes from a cytoskeletal type of pattern to a perinuclear localization within activated HSCs. Within 24 h of heat recovery, the cellular distribution of alpha B-crystallin returns to its normal localization pattern. A similar redistribution response for alpha B-crystallin after heat shock has been described in other cell types (8, 10). This response is believed to represent molecular chaperone activity in protecting cellular proteins from heat stress.

alpha B-crystallin along with alpha A-crystallin constitute the subunit proteins for alpha -crystallin. alpha -Crystallin represents >30% of the major soluble protein in vertebrate eye lens (8). Expression of alpha B-crystallin is generally restricted to the eye lens; however, low levels of expression have been reported in the spleen, thymus, and retina (30, 51, 56). alpha B-crystallin, on the other hand, is expressed in a variety of tissues, including the heart, skeletal muscle, kidney, placenta, skin, brain, peripheral nerves, spinal cord, retina, and liver (8, 16, 20, 21, 36). On the basis of structural and nucleotide sequence homology, alpha B-crystallin belongs to the small heat shock protein family, which includes the heat shock proteins HSP20, HSP25, HSP27, and HSP30 (8, 32). We compared the expression pattern of alpha B-crystallin with the small heat shock protein HSP25 and with the classic heat shock protein HSP70 in quiescent and culture-activated HSCs and after heat shock in activated HSCs. The expression pattern was similar between the small heat shock proteins alpha B-crystallin and HSP25. Both proteins were not expressed in quiescent HSCs, but expression was induced after HSC activation. Following heat shock, protein levels of these small heat shock proteins did not appreciably increase. In contrast, expression of HSP70 was not detected in quiescent or activated HSCs. HSP70 mRNA expression was found to be transiently increased after heat shock, although HSP70 protein levels remained elevated 24 h after the heat shock. Therefore, expression of small heat shock proteins differs from the classic heat shock protein HSP70 in HSCs. Although others (16, 32) have reported that alpha B-crystallin is induced after heat treatment, we did not observe heat induced alpha B-crystallin expression in activated HSCs. Constitutive expression of alpha B-crystallin has been reported in other cell types in the absence of stress (31). It is possible that the level of expression within the activated HSC is at maximum expression and may not be able to be further increased. Because HSCs are generally activated during stress, as seen in the oxidative stress conditions that occur during chronic alcohol consumption, the cell may be preprogrammed to cope with continued stress by constitutively expressing the small heat shock proteins, including alpha B-crystallin and HSP25. HSP70 expression may be reserved for dealing with additional, transient stress conditions to which the cell may be exposed. Alternatively, the different classes of heat shock proteins may serve to protect cells from different types of stress conditions.

Increased expression of alpha B-crystallin has been shown to be induced by high oxidative activity and thermal and osmotic stress and following treatment of cells with tumor necrosis factor-alpha (TNF-alpha ) (7, 12, 20, 39). We found alpha B-crystallin expression to be induced early in culture. Cultured HSCs are exposed to higher oxidative conditions than these cells normally are exposed to in the liver. This may explain the early and high expression levels of alpha B-crystallin when these cells are cultured. Both bile duct ligation and alcoholic liver disease represent high oxidative stress conditions in the liver during which alpha B-crystallin expression has also been shown to increase (Fig. 6) (16). Interestingly, administration of ethanol before but not during stress in rats can inhibit the increased stress-induced alpha B-crystallin expression (16).

Phosphorylation of alpha B-crystallin increases after stress (17) when specific serine residues are phosphorylated by both cAMP-dependent (50, 53) and cAMP-independent mechanisms (27). The p38 mitogen-activated protein kinase (MAPK) (29) as well as p44/p42 MAPK and MAPK-activated protein kinase 2 (28) have been shown to phosphorylate alpha B-crystallin. alpha B-crystallin has also been reported (25, 26) to possess an autokinase activity. Interestingly, we did not detect serine phosphorylation of alpha B-crystallin in culture-activated HSCs (Fig. 5). Evidently, the stress conditions of culture are not sufficient to induce phosphorylation of alpha B-crystallin. The biological effects of phosphorylation are poorly understood. It has been shown (55) that phosphorylation decreases oligomer size; however, the consequence of phosphorylation does not affect chaperone activity of the protein measured by suppression of thermal-induced aggregation of low B- or gamma -crystallins.

Heat shock proteins are implicated in a variety of medical conditions, including ischemia, stroke, cardiovascular disease, cancer, inflammation, trauma, aging, and autoimmunity, among others (5, 21-23, 35, 36, 41). alpha B-crystallin has been shown (4) to protect cells against ultraviolet-induced protein aggregation. The small heat shock proteins, including alpha B-crystallin, have also been shown to confer thermoresistance to cells (19) and provide protection against TNF-alpha (39), oxidative stress (39), and ischemic injuries (9). These proteins have also been shown (40) to protect cells against apoptosis mediated by staurosporine and Fas ligand. Resistance to TNF-alpha -mediated cell death is at least partially due to increased glutathione levels (1). Small heat shock proteins have been shown (1) to increase cell survival due to oxidative stress by decreasing intracellular reactive oxygen species in a glutathione-dependent manner. The small heat shock proteins also inhibit protease activity (44). This may be useful to prevent endogenous cellular proteases from attacking partially or transiently denatured proteins during stress.

Associations of alpha B-crystallin have been observed with several cytoskeletal components, including actin (6, 34, 56), desmin (2, 34), peripherin (10), vimentin (10, 45), and GFAP (45). It is believed that these associations protect against ischemic stress conditions and heat-induced protein aggregation in a chaperone-like manner. In fact, alpha B-crystallin inhibits heat-induced aggregation of actin (17). The molecular chaperone activity of the small heat shock proteins is more pronounced at increased temperatures (10, 46). It is possible that we did not observe alpha B-crystallin colocalization with the cytoskeletal proteins we examined since our studies were performed at normal, unstressed temperatures. The small heat shock proteins have also been shown to associate with membranes (15).

The demonstration that expression of both HSP25 and alpha B-crystallin increases after HSC activation represents two additional heat shock proteins whose expression is induced following HSC activation. Expression of HSP45, a collagen-specific molecular chaperone, has been also been shown to increase after HSC activation. HSP45 synthesis parallels collagen synthesis during normal development in a tissue-specific, temporal, and spatial manner (38). In addition, HSP45 gene expression is increased after a fibrogenic stimulus. HSP45 gene expression follows the increased expression of types I and III collagen and is expressed in the activated HSC (37, 42). Our study demonstrates that not all heat shock proteins are similarly regulated after HSC activation. Whereas expression of alpha B-crystallin, HSP25, and HSP45 is increased after HSC activation, expression of HSP70 is not increased during activation but is induced following heat shock in the activated HSC. This suggests significantly different roles for these stress proteins in the cell. HSP45 has been shown to be a collagen-specific molecular chaperone; however, the roles of HSP25 and alpha B-crystallin in the HSC remain unknown. Analysis of the functions of these proteins in other cell types suggest that these proteins probably act as molecular chaperones protecting the HSC from cellular stress.


    ACKNOWLEDGEMENTS

This work was supported by National Institute on Alcohol Abuse and Alcoholism Grants AA-10459 (R. A. Rippe) and AA-06603 (H. Tsukamoto), National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-34987 (R. A. Rippe and D. A. Brenner), and the Tissue Culture Core Facility of the University of Southern California Research Center for Liver Disease (Grant P30-DK-48522).


    FOOTNOTES

Address for reprint requests and other correspondence: R. A. Rippe, CB# 7038, Division of Digestive Diseases, Dept. of Medicine, Univ. of North Carolina, Chapel Hill, NC 27599-7038.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1 February 2000; accepted in final form 29 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Arrigo, AP. Small stress proteins: chaperones that act as regulators of intracellular redox state and programmed cell death. Biol Chem 379: 19-26, 1998[ISI][Medline].

2.   Bennardini, F, Wrzosek A, and Chiesi M. Alpha B-crystallin in cardiac tissue. Association with actin and desmin filaments. Circ Res 71: 288-294, 1992[Abstract].

3.   Bhat, SP, Horwitz J, Srinivasan A, and Ding L. AlphaB-crystallin exists as an independent protein in the heart and in the lens. Eur J Biochem 102: 775-781, 1991.

4.   Borkman, RF, Knight G, and Obi B. The molecular chaperone alpha-crystallin inhibits UV-induced protein aggregation. Exp Eye Res 62: 141-148, 1996[ISI][Medline].

5.   Chiesi, M, and Bennardini F. Determination of alpha B crystallin aggregation: a new alternative method to assess ischemic damage of the heart. Basic Res Cardiol 87: 38-46, 1992[ISI][Medline].

6.   Chiesi, M, Longoni S, and Limbruno U. Cardiac alpha-crystallin. III. Involvement during heart ischemia. Mol Cell Biochem 97: 129-136, 1990[ISI][Medline].

7.   Dasgupta, S, Hohman TC, and Carper D. Hypertonic stress induces alpha B-crystallin expression. Exp Eye Res 54: 461-470, 1992[ISI][Medline].

8.   de Jong, WW, Leunissen JA, and Voorter CE. Evolution of the alpha-crystallin/small heat-shock protein family. Mol Biol Evol 10: 103-126, 1993[Abstract].

9.   Dillmann, WH. Small heat shock proteins and protection against injury. Ann NY Acad Sci 874: 66-68, 1999[Abstract/Free Full Text].

10.   Djabali, K, de Nechaud B, Landon F, and Portier MM. AlphaB-crystallin interacts with intermediate filaments in response to stress. J Cell Sci 110: 2759-2769, 1997[Abstract/Free Full Text].

11.   Friedman, SL. Hepatic stellate cells. Prog Liver Dis 14: 101-130, 1996[Medline].

12.   Graw, J. The crystallins: genes, proteins and diseases. Biol Chem 378: 1331-1348, 1997[ISI][Medline].

13.   Gunning, P, Leavitt J, Muscat G, Ng SY, and Kedes L. A human beta-actin expression vector system directs high-level accumulation of antisense transcripts. Proc Natl Acad Sci USA 84: 4831-4835, 1987[Abstract].

14.   Hellerbrand, C, Wang SC, Tsukamoto H, Brenner DA, and Rippe RA. Expression of intercellular adhesion molecule 1 by activated hepatic stellate cells. Hepatology 24: 670-676, 1996[Medline].

15.   Ifeanyi, F, and Takemoto L. Specificity of alpha crystallin binding to the lens membrane. Curr Eye Res 9: 259-265, 1990[ISI][Medline].

16.   Inaguma, Y, Hasegawa K, Goto S, Ito H, and Kato K. Induction of the synthesis of hsp27 and alpha B crystallin in tissues of heat-stressed rats and its suppression by ethanol or an alpha 1-adrenergic antagonist. J Biochem (Tokyo) 117: 1238-1243, 1995[Abstract].

17.   Ito, H, Okamoto K, Nakayama H, Isobe T, and Kato K. Phosphorylation of alpha B-crystallin in response to various types of stress. J Biol Chem 272: 29934-29941, 1997[Abstract/Free Full Text].

18.   Iwaki, A, Iwaki T, Goldman JE, and Liem RK. Multiple mRNAs of rat brain alpha-crystallin B chain result from alternative transcriptional initiation. J Biol Chem 265: 22197-22203, 1990[Abstract/Free Full Text].

19.   Iwaki, T, Iwaki A, Tateishi J, and Goldman JE. Sense and antisense modification of glial alpha B-crystallin production results in alterations of stress fiber formation and thermoresistance. J Cell Biol 125: 1385-1393, 1994[Abstract].

20.   Iwaki, T, Kume-Iwaki A, and Goldman JE. Cellular distribution of alpha B-crystallin in non-lenticular tissues. J Histochem Cytochem 38: 31-39, 1990[Abstract].

21.   Iwaki, T, Kume-Iwaki A, Liem RK, and Goldman JE. Alpha B-crystallin is expressed in non-lenticular tissues and accumulates in Alexander's disease brain. Cell 57: 71-78, 1989[ISI][Medline].

22.   Iwaki, T, and Tateishi J. Immunohistochemical demonstration of alphaB-crystallin in hamartomas of tuberous sclerosis. Am J Pathol 139: 1303-1308, 1991[Abstract].

23.   Iwaki, T, Wisniewski T, Iwaki A, Corbin E, Tomokane N, Tateishi J, and Goldman JE. Accumulation of alpha B-crystallin in central nervous system glia and neurons in pathologic conditions. Am J Pathol 140: 345-356, 1992[Abstract].

24.   Jakob, U, Gaestel M, Engel K, and Buchner J. Small heat shock proteins are molecular chaperones. J Biol Chem 268: 1517-1520, 1993[Abstract/Free Full Text].

25.   Kantorow, M, Horwitz J, van Boekel MA, de Jong WW, and Piatigorsky J. Conversion from oligomers to tetramers enhances autophosphorylation by lens alpha A-crystallin. Specificity between alpha A- and alpha B-crystallin subunits. J Biol Chem 270: 17215-17220, 1995[Abstract/Free Full Text].

26.   Kantorow, M, and Piatigorsky J. Alpha-crystallin/small heat shock protein has autokinase activity. Proc Natl Acad Sci USA 91: 3112-3116, 1994[Abstract].

27.   Kantorow, M, and Piatigorsky J. Phosphorylations of alpha A- and alpha B-crystallin. Int J Biol Macromol 22: 307-314, 1998[ISI][Medline].

28.   Kato, K, Ito H, Kamei K, Inaguma Y, Iwamoto I, and Saga S. Phosphorylation of alphaB-crystallin in mitotic cells and identification of enzymatic activities responsible for phosphorylation. J Biol Chem 273: 28346-28354, 1998[Abstract/Free Full Text].

29.   Kato, K, Ito H, Kamei K, and Iwamoto I. Selective stimulation of Hsp27 and alphaB-crystallin but not Hsp70 expression by p38 MAP kinase activation. Cell Stress Chaperones 4: 94-101, 1999[ISI][Medline].

30.   Kato, K, Shinohara H, Kurobe N, Goto S, Inaguma Y, and Ohshima K. Immunoreactive alpha A crystallin in rat non-lenticular tissues detected with a sensitive immunoassay method. Biochim Biophys Acta 1080: 173-180, 1991[ISI][Medline].

31.   Klemenz, R, Andres A-C, Frohli E, Schafer R, and Aoyama A. Expression of the murine small heat shock proteins hsp 25 and alpha B crystallin in the absence of stress. J Cell Biol 120: 639-645, 1993[Abstract].

32.   Klemenz, R, Frohli E, Steiger RH, Schafer R, and Aoyama A. Alpha B-crystallin is a small heat shock protein. Proc Natl Acad Sci USA 88: 3652-3656, 1991[Abstract].

33.   Liang, P, and Pardee AB. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257: 967-971, 1992[ISI][Medline].

34.   Longoni, S, Lattonen S, Bullock G, and Chiesi M. Cardiac alpha-crystallin. II. Intracellular localization. Mol Cell Biochem 97: 121-128, 1990[ISI][Medline].

35.   Lowe, J, Landon M, Pike I, Spendlove I, McDermott H, and Mayer RJ. Dementia with beta-amyloid deposition: involvement of alpha B-crystallin supports two main diseases. Lancet 336: 515-516, 1990[ISI][Medline].

36.   Lowe, J, McDermott H, Pike I, Spendlove I, Landon M, and Mayer RJ. Alpha B crystallin expression in non-lenticular tissues and selective presence in ubiquitinated inclusion bodies in human disease. J Pathol 166: 61-68, 1992[ISI][Medline].

37.   Masuda, H, Fukumoto M, Hirayoshi K, and Nagata K. Coexpression of the collagen-binding stress protein HSP47 gene and the alpha 1(I) and alpha 1(III) collagen genes in carbon tetrachloride-induced rat liver fibrosis. J Clin Invest 94: 2481-2488, 1994[ISI][Medline].

38.   Masuda, H, Hosokawa N, and Nagata K. Expression and localization of collagen-binding stress protein Hsp47 in mouse embryo development: comparison with types I and II collagen. Cell Stress Chaperones 3: 256-264, 1998[ISI][Medline].

39.   Mehlen, P, Preville X, Chareyron P, Briolay J, Klemenz R, and Arrigo AP. Constitutive expression of human hsp27, Drosophila hsp27, or human alpha B-crystallin confers resistance to TNF- and oxidative stress-induced cytotoxicity in stably transfected murine L929 fibroblasts. J Immunol 154: 363-374, 1995[Abstract/Free Full Text].

40.   Mehlen, P, Schulze-Osthoff K, and Arrigo AP. Small stress proteins as novel regulators of apoptosis. Heat shock protein 27 blocks Fas/APO-1- and staurosporine-induced cell death. J Biol Chem 271: 16510-16514, 1996[Abstract/Free Full Text].

41.   Murano, S, Thweatt R, Shmookler Reis RJ, Jones RA, Moerman EJ, and Goldstein S. Diverse gene sequences are overexpressed in Werner syndrome fibroblasts undergoing premature replicative senescence. Mol Cell Biol 11: 3905-3914, 1991[ISI][Medline].

42.   Nagata, K. Expression and function of heat shock protein 47: a collagen-specific molecular chaperone in the endoplasmic reticulum. Matrix Biol 16: 379-386, 1998[ISI][Medline].

43.   Ohata, M, Lin M, Satre M, and Tsukamoto H. Diminished retinoic acid signaling in hepatic stellate cells in cholestatic liver fibrosis. Am J Physiol Gastrointest Liver Physiol 272: G589-G596, 1997[Abstract/Free Full Text].

44.   Ortwerth, BJ, and Olesen PR. Characterization of the elastase inhibitor properties of alpha-crystallin and the water-insoluble fraction from bovine lens. Exp Eye Res 54: 103-111, 1992[ISI][Medline].

45.   Perng, MD, Cairns L, van den IJssel P, Prescott A, Hutcheson AM, and Quinlan RA. Intermediate filament interactions can be altered by HSP27 and alphaB-crystallin. J Cell Sci 112: 2099-2112, 1999[Abstract/Free Full Text].

46.   Raman, B, Ramakrishna T, and Rao CM. Temperature dependent chaperone-like activity of alpha-crystallin. FEBS Lett 365: 133-136, 1995[ISI][Medline].

47.   Ratziu, V, Lalazar A, Wong L, Dang Q, Collins C, Shaulian E, Jensen S, and Friedman SL. Zf9, a Kruppel-like transcription factor up-regulated in vivo during early hepatic fibrosis. Proc Natl Acad Sci USA 95: 9500-9505, 1998[Abstract/Free Full Text].

48.   Rippe, RA, Almounajed G, and Brenner DA. Sp1 binding activity increases in activated Ito cells. Hepatology 22: 241-251, 1995[ISI][Medline].

49.   Rippe, RA, Schrum LW, Stefanovic B, Solís-Herruzo JA, and Brenner DA. NF-kB inhibits expression of the alpha 1(I) collagen gene. DNA Cell Biol 18: 751-761, 1999[ISI][Medline].

50.   Spector, A, Chiesa R, Sredy J, and Garner W. cAMP-dependent phosphorylation of bovine lens alpha-crystallin. Proc Natl Acad Sci USA 82: 4712-4716, 1985[Abstract].

51.   Srinivasan, AN, Nagineni CN, and Bhat SP. Alpha A-crystallin is expressed in non-ocular tissues. Biol Chem 267: 23337-23341, 1992[Abstract/Free Full Text].

52.   Tsukamoto, H, Cheng S, and Blaner WS. Effects of dietary polyunsaturated fat on ethanol-induced Ito cell activation. Am J Physiol Gastrointest Liver Physiol 270: G581-G586, 1996[Abstract/Free Full Text].

53.   Voorter, CE, Mulders JW, Bloemendal H, and de Jong WW. Some aspects of the phosphorylation of alpha-crystallin A. Eur J Biochem 160: 203-210, 1986[Abstract].

54.   Wan, Y-J, Wang L, and Wu T-CJ. Detection of retinoic acid receptor mRNA in rat tissues by reverse transcriptase-polymerase chain reaction. J Mol Endocrinol 9: 291-294, 1992[Abstract].

55.   Wang, K, Ma W, and Spector A. Phosphorylation of alpha-crystallin in rat lenses is stimulated by H2O2 but phosphorylation has no effect on chaperone activity. Exp Eye Res 61: 115-124, 1995[ISI][Medline].

56.   Wang, K, and Spector A. alpha -Crystallin stabilizes actin filaments and prevents cytochalasin-induced depolymerization in a phosphorylation-dependent manner. Eur J Biochem 242: 56-66, 1996[Abstract].


Am J Physiol Gastrointest Liver Physiol 279(6):G1333-G1342
0193-1857/00 $5.00 Copyright © 2000 the American Physiological Society