Search for a functional glucocorticoid receptor in the mammalian lens.

Prolonged glucocorticoid treatment of medical conditions such as rheumatoid arthritis or asthma can lead to the formation of a posterior subcapsular cataract as a negative side effect. Currently, the only treatment for this cataract is surgery because very little is known about the mechanism of glucocorticoid action in the mammalian lens. Understanding of a lens glucocorticoid response is essential for the treatment and prevention of a steroid induced cataract. It has been suggested that glucocorticoids exert their effects on the lens indirectly, non-specifically, or through non-classical mechanisms. While these modes of action may contribute to the formation of glucocorticoid induced posterior subcapsular cataract, the finding of a classical, specific, functional lens glucocorticoid receptor suggests that glucocorticoids target lens epithelial cells directly, specifically, and similar to what has been observed in other cells types. This review explores the discovery of the glucocorticoid receptor in humans lens epithelial cells and the lens specific glucocorticoid response. The distinct changes in lens epithelial cell signaling pathways (MAPK and PI3K-AKT) suggest that glucocorticoids modulate several cellular functions and may explain why a lens glucocorticoid response has been difficult to elucidate.

Downregulation of MMP-2 and -9 by proteasome inhibition: a possible mechanism to decrease LEC migration and prevent posterior capsular opacification.

PURPOSE: The proliferation, epithelial-mesenchymal transition (EMT), and migration of residual lens epithelial cells (LECs) after cataract surgery leads to the development of posterior capsular opacification (PCO). The authors have shown that proteasome inhibition suppresses LEC proliferation and EMT. The present study investigates the prevention of LEC migration by proteasome inhibition through the suppression of matrix metalloproteinase (MMP) expression and activity. METHODS: HLE B-3 and primary human LEC migration assays were performed using polycarbonate membrane inserts and 20% fetal bovine serum (FBS) as chemoattractant. Cultured cells were treated with 1 ng TGF-beta(2), with or without MG132 (proteasome inhibitor) or GM 6001 (MMP inhibitor). Capsular bags with intraocular lenses (IOLs) were prepared from human donor eyes and cultured in serum-free DMEM. The capsular bags were then treated with 1 or 10 ng/mL TGF-beta(2), with or without MG132 (2.5 or 10 muM, respectively). The medium was sampled and replaced every 2 days and analyzed for MMP-2 and -9 activities by SDS-PAGE zymography. Protein and RNA expression were analyzed by Western blotting and RT-PCR, respectively. RESULTS: Proteasome inhibition blocks LEC migration in HLE B-3 and primary human LECs. To further evaluate the mechanism of decrease in LEC migration by proteasome inhibition, the authors measured MMP-2 mRNA and protein expression and MMP-2 and -9 activities. In HLE B-3 cells, TGF-beta(2) increased MMP-2 mRNA and protein levels; these increases were inhibited by MG132 cotreatment. Medium from HLE B-3 cultures showed MMP-2 and -9 activities, which were induced by TGF-beta(2) treatment and inhibited by MG132 co-treatment. TGF-beta(2) treatment also increased MMP-2 and -9 activities in IOL capsular bag cultures; these were progressively decreased by proteasome inhibition. CONCLUSIONS: Proteasome inhibition decreases LEC migration. This inhibition is correlated with decreased MMP-2 and -9 activities, observed both with and without TGF-beta(2) treatment. These findings support proteasome inhibition as a therapeutic strategy to prevent PCO.

Specific activation of the glucocorticoid receptor and modulation of signal transduction pathways in human lens epithelial cells.

PURPOSE: Prolonged use of glucocorticoids (GCs) can lead to cataract formation. Lens GC responses have been difficult to elucidate. A previous study showed the presence of the glucocorticoid receptor (GR) in immortalized and primary human lens epithelial cells (hLECs) and GC-induced changes in gene expression. This study demonstrates specific GR activation and identifies the biological effect of GC-induced changes in gene expression in hLECs. METHODS: HLE B-3 (B-3) and primary cultures of hLECs were transfected with pGRE.Luc and treated with or without dexamethasone (Dex), RU-486, spironolactone, or vehicle. mRNA and protein expression were examined by real-time PCR and Western blot analysis, respectively. Cell proliferation and apoptosis were examined by WST-1 and flow cytometry, respectively. RESULTS: Dex treatment of B-3 and primary cultures demonstrated specific GR, but not mineralocorticoid receptor (MR), activation and phosphorylation. Pathway analysis revealed GC-induced changes in expression of MAPK regulators. Increased expression of GILZ mRNA and MKP-1 mRNA and protein was observed in immortalized and donor hLECs. This corresponded with a decrease in the phosphorylated forms of RAF, ERK, p38, and AKT, but not in JNK. No net change in LEC proliferation or apoptosis was observed with Dex treatment. CONCLUSIONS: GC treatment of hLECs activates the GR to modulate the expression of MAPK and PI3K/AKT regulators. This is the first demonstration of GC signaling in hLECs. GCs, MAPK, and PI3K/AKT are involved in cell processes implicated in steroid-induced cataractogenesis. The absence of a net change in cell activity with acute steroid treatment is consistent with the possibility that chronic treatment leads to prolonged modulation of these pathways and steroid-induced cataract.

Purification, characterization, and cDNA cloning of an AU-rich element RNA-binding protein, AUF1.

The degradation of some proto-oncogene and lymphokine mRNAs is controlled in part by an AU-rich element (ARE) in the 3' untranslated region. It was shown previously (G. Brewer, Mol. Cell. Biol. 11:2460-2466, 1991) that two polypeptides (37 and 40 kDa) copurified with fractions of a 130,000 x g postribosomal supernatant (S130) from K562 cells that selectively accelerated degradation of c-myc mRNA in a cell-free decay system. These polypeptides bound specifically to the c-myc and granulocyte-macrophage colony-stimulating factor 3' UTRs, suggesting they are in part responsible for selective mRNA degradation. In the present work, we have purified the RNA-binding component of this mRNA degradation activity, which we refer to as AUF1. Using antisera specific for these polypeptides, we demonstrate that the 37- and 40-kDa polypeptides are immunologically cross-reactive and that both polypeptides are phosphorylated and can be found in a complex(s) with other polypeptides. Immunologically related polypeptides are found in both the nucleus and the cytoplasm. The antibodies were also used to clone a cDNA for the 37-kDa polypeptide. This cDNA contains an open reading frame predicted to produce a protein with several features, including two RNA recognition motifs and domains that potentially mediate protein-protein interactions. These results provide further support for a role of this protein in mediating ARE-directed mRNA degradation.

Suppression of human lens epithelial cell proliferation by proteasome inhibition, a potential defense against posterior capsular opacification.

PURPOSE: Posterior capsular opacification (PCO) is caused by the proliferation, migration, and epithelial-mesenchymal transition (EMT) of the remaining lens epithelial cells (LECs) after cataract surgery. Studies have shown that proteasome inhibition interferes with EMT and remodeling of the extracellular matrix. This study was conducted to investigate suppression of LEC proliferation by proteasome inhibition and its signaling pathway. METHODS: HLE B-3 cells and human lens epithelium explants from 17- to 20-week fetal lenses were cultured and treated with TGF-beta2 (1 or 10 ng/mL), FGF-2 (20 or 50 ng/mL), HGF (10 ng/mL) and 5 or 10 muM MG132. LEC proliferation was determined using both the WST-1 reagent and proliferating cell nuclear antigen (PCNA) expression. Protein expression was observed by Western blot analysis. Transfection with p21/p27 siRNA was performed to evaluate the mechanism of the antiproliferative effect of proteasome inhibition. RESULTS: TGF-beta2 suppressed proliferation of HLE B-3 cells, whereas FGF-2 and HGF enhanced proliferation. Proliferation suppression by TGF-beta2 was blocked by adding FGF-2 or HGF. Proteasome inhibitor (MG132) treatment strongly inhibited the proliferation of LECs, either alone or in the presence of TGF-beta2, FGF-2, or HGF. These findings were confirmed by observing PCNA expression. Similar results were obtained with primary human LECs. Expression of cell cycle regulatory proteins was determined to evaluate the mechanism of the antiproliferative activity of proteasome inhibition. MG132 caused a significant increase in p21 and p27 protein and decrease in CDK2, but no change in p53, p57, CDK4, or CDK6 protein. The antiproliferative effect of MG132 was significantly reversed in samples transfected with p21 and p27 siRNA, which reduced p21 and p27 protein expression to very low levels that remained below basal control levels, even after treatment with MG132. CONCLUSIONS: Proteasome inhibition decreases the proliferation of LECs in the presence or absence of TGF-beta2, FGF-2, and HGF. This process is mediated in part by an increase in p21 and p27 proteins. These findings suggest that proteasome inhibitors are good candidates for blocking development of PCO.

Role of the proteasome in TGF-beta signaling in lens epithelial cells.

PURPOSE: The durability of the ubiquitin proteasome pathway in the mammalian lens makes this enzyme system a potential contributor to certain cataracts and posterior capsular opacification (PCO). The present study addresses proteasome involvement in TGF-beta induced, cataract-associated gene activation in human lens cells. METHODS: HLE B-3 cells were treated with TGF-beta, in combination with the proteasome inhibitors MG-132 or lactacystin. TGF-beta target gene expression was measured by semiquantitative RT-PCR. Annexin-FITC staining and flow cytometry were used to assess apoptosis levels. Western blot analyses were performed with anti-SnoN and anti-Smad2 antibodies. RESULTS: TGF-beta induced the expression of alpha-smooth muscle actin, fibronectin, and TGF-beta-inducible gene mRNA in HLE B-3 cells and primary cultured human lens cells from donor tissues. TGF-beta also induced a time-dependent decrease in the level of the Smad repressor SnoN. Gamma-glutamyl-cysteine synthetase (gamma-GCS) mRNA levels decreased in the presence of TGF-beta. Proteasome inhibitor cotreatment blocked the induction of alpha-SMA mRNA, the loss of SnoN protein, the decrease in gamma-GCS mRNA, and TGF-beta-induced apoptosis. CONCLUSIONS: The HLE B-3 cell line and primary cultured human lens cells respond similarly to TGF-beta treatments by activating cataract-related gene expression. This response in both of these model systems is blocked by inhibiting the proteasome. This suggests that the proteasome can mediate cataract and PCO-associated changes and therefore is a novel target of medical therapy.

Upregulation of heat shock protein expression by proteasome inhibition: an antiapoptotic mechanism in the lens.

PURPOSE: Studies have shown that proteasome inhibition protects lens epithelial cells (LECs) against interferon (IFN)-gamma-induced apoptosis. The present study was conducted to test the hypothesis that proteasome inhibition can protect lens cells against apoptosis by upregulating heat shock protein (HSP) expression. METHODS: Murine lens epithelial alphaTN4-1 cells were treated with combinations of 100 U/mL IFN-gamma, 10 muM MG132 (proteasome inhibitor), and 100 muM quercetin (HSP inhibitor). mRNA and protein expression were observed by RT-PCR and Western blot analysis, respectively. Caspase activities were measured by using cleavage of colorimetric substrate. Apoptosis was measured by phase-contrast microscopy and flow cytometry. RESULTS: At the mRNA level, the proteasome inhibitor, MG132, caused a >10-fold increase in HSP27 and a small increase (1.2- to 1.6-fold) in alphaB-crystallin but no change in HSP70 or -90. At the protein level, a more than twofold increase in HSP27 and -90, a marked increase in HSP70, but no significant change in alphaB-crystallin, was observed. Downregulation of alphaA-crystallin by MG132 was observed at both the mRNA and protein levels. MG132 caused no significant change in heat shock factor (HSF)-1, but a more than twofold increase in HSF2 and -4 protein expression. MG132 prevented the IFN-gamma-induced increase in caspase-1, -6, and -8 activities. Quercetin decreased MG132-induced expression of HSP27, -70, and -90 by more than 70%, and heat shock factors HSF2 and -4 by more than 65%. Quercetin pretreatment significantly reversed the decrease in caspase-1, -6, and -8 activities and the antiapoptotic effect of MG132 on IFN-gamma-treated LECs. CONCLUSIONS: The antiapoptotic effect of proteasome inhibition of IFN-gamma-induced apoptosis in LECs correlates with increased expression of HSPs and inhibition of caspase activities. Inhibition of HSP expression restores caspase activities and abolishes the antiapoptotic effect of proteasome inhibition, implicating HSPs as mediators of the protective effect of proteasome inhibition.

Global gene profiling reveals novel glucocorticoid induced changes in gene expression of human lens epithelial cells.

PURPOSE: Prolonged use of glucocorticoids can lead to the formation of a cataract, however the mechanism is not known. We recently reported the presence of the functional glucocorticoid receptor in immortalized cultured mammalian lens epithelial cells (LECs), but the biological effect is not known. This study seeks to determine if freshly isolated human LECs respond to glucocorticoid treatment and to examine glucocorticoid induced changes in global gene expression in LECs. METHODS: Capsulorhexis specimens obtained in surgery from eyes with cataract were cultured. Primary lens cultures were transfected, in triplicate, with pGRE.Luc, which drives the expression of firefly luciferase, and treated with dexamethasone (Dex) or vehicle (Veh). RNA isolated from HLE B-3 cells, treated with Dex or Veh for 4 or 16 h in triplicate, was used to analyze global changes in gene expression by microarray hybridization. Data and cluster analyses were performed using Microarray Suite 5.0, GeneSpring 6.1, EASE, NetAffx, and SAM. Real Time PCR was used to confirm microarray data in RNA isolated from HLE B-3 cells in triplicate and a primary culture of human lens epithelial cells. RESULTS: Transfected primary cultures of human LECs treated with Dex demonstrated a glucocorticoid response with a greater than 4 fold increase in firefly luciferase activity over controls. Microarray data revealed that 136 genes were modulated with 4 h treatment with Dex. Of the 136 genes, 93 transcripts were upregulated and 43 were downregulated by greater than 1.5 fold. Eighty-six genes were modulated with 16 h Dex treatment. Of the 86 genes, 30 transcripts were upregulated and 56 were downregulated by greater than 1.5 fold. Microarray results were verified by Real Time PCR in both the HLE B-3 and primary cultures of lens epithelial cell. CONCLUSIONS: The activation of a GRE reporter gene in primary cultures of human LECs demonstrates that the glucocorticoid receptor is functional in non-immortalized human lens cells. Microarray studies at 2 time periods demonstrate that glucocorticoids modulate gene expression in immortalized human LECs, reveal novel changes in gene expression, and confirm an endogenous genomic lens glucocorticoid response. This study demonstrates that primary cultures of lens epithelial cells and microarray technology can be used to determine pathways involved in a lens glucocorticoid response and lead to a better understanding of the formation of a steroid induced cataract.

Targeted disruption of specific steps of the ubiquitin-proteasome pathway by oxidation in lens epithelial cells.

Several steps in the ubiquitin-proteasome pathway have been shown to be inhibited in models of oxidative stress and aging. We have designed similar models of aging and oxidation in the HLE B-3 human lens epithelial cell line. Following hydrogen peroxide (H2O2) treatment, B-3 cells exhibited an expected activation of c-fos. The effect of these same and similar treatments on the lens proteasome system was unexpected. The 2D gel pattern and the chymotrypsin-like activity of the 20S core were unaffected by this H2O2 treatment, contrary to previous experience in other culture systems. The critical role of proteolysis in the aging lens, and the strong tie between oxidation and proteasome changes, urged us to further model lens oxidation and investigate several steps of the ubiquitin-proteasome pathway with an alternative agent: the thiol-specific oxidant, diamide. The 20S core proteasome, de-ubiquitinating, and ATP-dependent 26S proteasome activities all showed decreases 10 min after diamide was applied, and recovered to near normal within 1h. The higher, 300 microM dose inhibited the 20S by 43%, the de-ubiquitinating activity by 17% and the 26S by 31%. The comparable susceptibility of the 20S activity and the 26S activity differs from several previously published models. Such differences may be the result of tissue or cell line-specific variants in either the components of the ubiquitin-proteasome pathway or in their modification by intracellular oxidants or reductants.

Characterization of cDNAs encoding the murine A+U-rich RNA-binding protein AUF1.

A+U-rich elements (ARE) serve to control the degradation of some proto-oncogene and lymphokine mRNAs. The protein, AUF1, which consists of two polypeptides of 37 and 40 kDa (p37 and p40, respectively) when purified from cytosol, has been implicated in ARE-directed mRNA turnover due to its binding to ARE. Molecular cloning of a cDNA (p37AUF1) corresponding to human p37 predicted a polypeptide containing two non-identical RNA recognition motifs (RRM) and a C-terminal Gln-rich domain [Zhang et al. Mol. Cell. Biol. 13 (1993) 7652-7665]. Two cDNAs, designated muAUF1-3 and muAUF1-7, were isolated from a murine fetal cDNA library, using as a probe, a fragment of the p37AUF1 cDNA encoding RRM1 and approximately half of RRM2. The muAUF1-3 open reading frame (ORF) was very homologous to human p37AUF1 with the greatest homology between the corresponding RRMs and the C-terminal Gln-rich motif. Clone muAUF1-7 was highly homologous to muAUF1-3, but was truncated within the region encoding the RNP-1 box in RRM2. Clone muAUF1-3 encoded 19 amino acids in RRM1 not encoded by either muAUF1-7 or human p37AUF1. Such alterations in sequence could modify the RNA-binding properties of these proteins and have concomitant effects on ARE-directed posttranscriptional processes.

Age changes in bovine lens endopeptidase activity.

Lens endopeptidase activity and thermal stability have been determined as a function of cell development, cell age, and animal age. Lenses from animals aged 3 months to 15 years (lens weights 1.15-2.80 g) were divided into epithelial (outermost), cortical (peripheral), and nuclear (central) regions. Changes accompanying cell development were determined by measuring specific activity in epithelial (undifferentiated), outer cortical (differentiating), inner cortical (mature) and nuclear (aged) regions of individual lenses. Thermal stability of the enzyme activity obtained from the outer cortical and nuclear regions of the same lenses was also determined. Specific activity and thermal stability were found to decrease as a function of lens cell development. Changes with cell development represent the effects of both differentiation and increasing cell age. To determine the effects of cell age alone, activity was determined in the same population of aged, fully differentiated cells in lenses of different ages. Specific activity decreased as a function of cell age alone. Changes with animal age were determined by comparing cells of the same developmental stage from animals of different ages (e.g., differentiating cells of the cortex in animals 3 months to 15 years old). Specific activity for the cortical region increased with animal age while specific activity in the nuclear region appeared to remain constant or decrease slightly with increasing animal age. Thermal stability of the enzyme activity from the cortex was different in young and adult lenses. The change in stability occurred early in the lifespan and was therefore more closely related to animal development than to aging.

Interferon-gamma induces apoptosis of lens alphaTN4-1 cells and proteasome inhibition has an antiapoptotic effect.

PURPOSE: Targeted ectopic expression of interferon-gamma (IFN-gamma) in the eyes of transgenic mice disrupts lens differentiation, upregulates immunoproteasomes, and causes cataract. In this study, the hypothesis that IFN-gamma induces proteasome-dependent apoptosis of lens epithelial cells was tested. METHOD: Murine lens epithelial alphaTN4-1 cells were treated with IFN-gamma. Apoptosis was measured using annexin V-FITC and propidium iodide (PI) staining, and DNA fragmentation. IFN-gamma-inducible mRNA and protein expressions were measured by RT-PCR and Western blot analysis. Caspase activities were measured using colorimetric substrates and poly (ADP ribose) polymerase (PARP) cleavage. The effect of proteasome inhibition was tested with MG132 and lactacystin. RESULTS: IFN-gamma treatment at a concentration that induces immunoproteasome expression causes an approximately 20% increase in early apoptotic cells as observed by annexin V-FITC/PI staining and the increase in DNA fragmentation. IFN-gamma-induced apoptosis was accompanied by upregulation of apoptosis-related genes, including a dramatic increase in signal transducer and activator of transcription (STAT)-1 and interferon consensus sequence binding protein (ICSBP), a more than 2-fold increase in IRF-1, and a 1.7- to 2-fold increase in caspase-1 mRNA. Bcl-2 mRNA decreased 2.4- to 3.0-fold, whereas Bax mRNA was unchanged. The Bax-to-Bcl-2 protein ratio increased by 1.6-fold. Caspase-1 and -8 activities were higher, but there was no increase in caspase-3 activity. Proteasome inhibitors MG132 and lactacystin protected the cells against IFN-gamma-induced apoptosis. A positive control treatment with staurosporine (STP) caused increased caspase-3 activity, which was inhibited by MG132. CONCLUSIONS: IFN-gamma causes apoptosis of alphaTN4-1 cells, accompanied by upregulation of known effectors. IFN-gamma-induced apoptosis involves Bcl-2 family proteins and caspases. Proteasome inhibition has antiapoptotic effects on IFN-gamma-induced apoptosis. It also inhibits the STP-induced increase in caspase-3 activity. If IFN-gamma-induced apoptosis of lens epithelial cells contributes to cataractogenesis, the proteasome may be a therapeutic target.

Expression of the functional glucocorticoid receptor in mouse and human lens epithelial cells.

PURPOSE: Studies have questioned the reported presence of a classic glucocorticoid receptor (GR) in the mammalian lens. The purpose of this study is to determine whether the functional GR is expressed in human and mouse lens epithelial cells. METHODS: GR mRNA was determined by RT-PCR in freshly isolated human lens epithelia, mouse lens, immortalized human (HLE B-3) and mouse (alphaTN4) lens epithelial cells and in mouse lung, NIH-3T3 cells, and HeLa cells, which served as positive controls. Western blot analysis with the GR-specific antibody H-300 was performed on protein extracts from human lens epithelia, HLE B-3 cells, and alphaTN4 cells and from HeLa cells, NIH-3T3 cells, and partially purified GR, which served as positive controls. pGRE.Luc drives the expression of firefly luciferase. HLE B-3 and alphaTN4 cells were transfected with pGRE.Luc and cotreated with dexamethasone, with and without the competitive inhibitor RU-486. RESULTS: PCR products of the expected size were detected in all samples, sequenced in both directions, and found to have 97% to 100% homology with the GR. A band in the appropriate molecular weight range was identified by Western blot analysis in the lens extracts. Active GR binding to the GRE was demonstrated by an increase in firefly luciferase expression in transfected cells treated with dexamethasone. The dexamethasone-induced increase in luciferase activity was inhibited with the addition of RU-486. CONCLUSIONS: These results demonstrate expression of the functional glucocorticoid receptor in mouse and human lens epithelial cells. This finding suggests that glucocorticoids may act on the mouse and human lens directly during normal lens development and/or cataractogenesis.

Proteome analysis of lens epithelia, fibers, and the HLE B-3 cell line.

PURPOSE: The purpose of this study is to compare the protein composition of the B-3 line of transformed human lens epithelial (HLE) cells to that of freshly dissected HLE cells. This provides baseline data on lens cell proteins from fresh lens cells and from the B-3 cell line, which is often used as a model system for the lens. METHODS: Human lens epithelial cells adherent to the lens capsule were dissected into central (undifferentiated) and peripheral (partially differentiated) populations. Fully differentiated human lens fiber cells were isolated from the outer cortical layers of the lens. HLE B-3 cells were analyzed at several passage levels. Extracts were prepared from each cell type and the proteins resolved by two-dimensional polyacrylamide gel electrophoresis (2-DE). Representative gel patterns were visually compared, spots excised, and trypsin digests prepared. The peptide compositions of the digests were analyzed using either liquid chromatography electrospray ionization tandem mass spectrometry or atmospheric pressure-matrix-assisted laser desorption ionization mass spectrometry, using a liquid chromatography classic ion trap (LCQ) mass spectrometer. RESULTS: Two-DE patterns were obtained for fresh and cultured cell types. Similar patterns were observed between central and peripheral HLE cells, both of which contained high levels of alphaA-, alphaB-, and betaB2-crystallins; alpha-enolase; and aldehyde dehydrogenase. HLE B-3 cultured cells were characterized by a marked loss of crystallins and a relatively higher level of noncrystallin proteins--most notably, high molecular weight, acidic proteins. Whereas subunit d of adenosine triphosphate (ATP) synthase, alphaB-crystallin, galectin, glyceraldehyde-3-phosphate dehydrogenase, alpha-enolase, actin, peptidylprolyl isomerase A, phosphatidylethanolamine-binding protein, and vimentin were present in both fresh and cultured lens epithelium, only the high abundance of alpha-enolase, galectin-1, and vimentin suggested that B-3 cells were lens derived. CONCLUSIONS: Freshly dissected noncultured HLE cells from both central and peripheral regions contain a high concentration of crystallins that mask the detection of less abundant proteins by 2-DE. Transformation and culture of HLE cells causes a loss of these crystallins and an increase in the relative concentration of other proteins. However, most of these noncrystallin proteins were different from those observed in noncultured HLE cells. These results suggest that transformation markedly alters the protein expression pattern in immortalized HLE cells and that caution should be exercised when using them to study properties of HLE cells in vivo.

Identification of a novel gene product preferentially expressed in rat lens epithelial cells.

PURPOSE: We searched for an mRNA that is differentially expressed in rat lens epithelial and fiber cells to use as a marker for differentiation. METHODS: An mRNA differential display method was used to identify genes with altered expression during differentiation of lens epithelial cells. RESULTS: One gene (partial sequence) was identified and confirmed to be expressed preferentially in undifferentiated rat lens epithelial cells. Among non-lens tissues, a trace amount was detected in cornea, but not in retina or eleven non-ocular tissues tested. This cloned partial gene sequence (322 base pairs) is at least 93% identical to overlapping rat and mouse EST sequences, and extends the sequence of both ESTs at the 5' end. CONCLUSIONS: A clone has been isolated that is preferentially expressed in lens epithelial cells. Expression of this gene was down-regulated when rat pup lens epithelial cell explants were induced to differentiate. The unusual pattern of expression suggests a novel function for this gene.

Covalent labelling of bovine lens multicatalytic proteinase complex with [3H]di-isopropyl fluorophosphate.

Enzymatically active lens multicatalytic proteinase complex bound [3H]iPr2P-F after incubation for 3 hours at ambient temperature. Label was associated with the lowest molecular weight band (Mr 22,000) on sodium dodecyl sulfate polyacrylamide gels. This binding was inhibited by preincubation of the enzyme with the cysteine-directed reagent, p-chloromercuribenzoate, which inhibits all three hydrolytic activities of the enzyme. Leupeptin, which inhibits the arginyl-hydrolyzing component, but not the iPr2P-F-inhibitable leucyl-hydrolyzing component of the enzyme, does not inhibit [3H]iPr2P-F binding. These data suggest that the leucy-hydrolyzing component of the lens multicatalytic proteinase complex is localized to the 22,000 Mr subunit and is a member of the thiol-dependent subclass of serine proteinases.

Conserved N-terminal sequences in homologous subunits of the multicatalytic proteinase complex (proteasome).

The bovine lens multicatalytic proteinase complex (MPC) (MW 700 kDa) comprises at least twelve subunits in the molecular mass range 22-35 kDa. Three of the subunits, L1 (27 kDa), L2 (24 kDa) and L3 (29 kDa), were purified by reverse phase HPLC. Their amino acid composition and N-terminal sequences indicate that they are not identical. L1 and L2 subunits show very high (greater than 90%) sequence homology with specific subunits of rat liver and human reticulocyte MPC and these are considered to be homologous components of the MPC which are highly conserved in evolution.

The bovine lens neutral proteinase comprises a family of cysteine-dependent proteolytic activities.

Inhibitor studies with peptide substrates demonstrate that bovine lens neutral proteinase comprises three distinct activities. Diisopropylfluorophosphate distinguishes the activity hydrolyzing carbobenzoxy-Gly-Gly-Leu-p-nitroanilide (inhibited) from that hydrolyzing carbobenzoxy-Leu-Leu-Glu-2-naphthylamide (not inhibited). Leupeptin inhibits hydrolysis of the substrate carbobenzoxy-Leu-Leu-Arg-2-naphthylamide, but not hydrolysis of carbobenzoxy-Gly-Gly-Leu-p-nitroanilide or carbobenzoxy-Leu-Leu-Glu-2-naphthylamide, demonstrating the presence of the third activity. Inhibition of the three activities by thiol reagents suggests that each activity may be dependent on an active-site cysteine residue.

The kidney: radiologic-pathologic correlation.

Selected concepts of MR-pathologic correlation of renal masses are presented in this article by way of illustrative examples, with an emphasis on renal adenocarcinoma. Where applicable, practical uses of these concepts are suggested, particularly in those cases in which the differential diagnosis might be narrowed, such as in the evaluation of angiomyolipoma. Also included is a discussion of management decisions that may be affected by the information provided by MR imaging.

The adrenal gland: radiologic-pathologic correlation.

The adrenal glands are affected by complex physiologic and neoplastic processes. Recently described computed tomography densitometry and chemical shift MR imaging techniques are useful to distinguish between benign and malignant masses. Knowledge of the basic gross pathologic and histologic correlates of adrenal disease helps in applying these new imaging methods and in understanding established cross-sectional studies.