Nonchromatin nuclear proteins of mammalian lens epithelial cells

The nuclear matrix (NM) proteins of six tissue cultured lens epithelial cell lines and one embryonic rabbit epidermal cell line were analyzed to determine possible tissue and species specificity of these proteins. The NM proteins were isolated by the modified Penman technique. The tissue cultured cells were pulsed with [³⁵S] methionine and nuclear matrix proteins were fractionated by two-dimensional (2-D) gel electrophoresis. The 2-D gels were dried and autoradiographed. The relative abundance of spot patterns of nuclear matrix proteins of different cells were compared. The data from these experiments revealed that all the examined cell lines have distinct spot patterns, however, all of NM profile showed a spot pattern in the 45 kDa region with acidic pH. Some of these spots cross-reacted with anti-vimentin antibodies, whereas a prominent protein spot in this region did not cross react with either vimentin or actin antibodies. The observed variations in the NM protein patterns of lens epithelial cells may reflect tissue and species specificity and also a role in the regulatory properties of these nuclear proteins in the eye tissue development. J. Cell. Biochem. 64:644–650. © 1997 Wiley-Liss, Inc.     

Intracellular protein degradation in cultured bovine lens epithelial cells

Summary Although several proteases have been identified in homogenates of cultured epithelial cells of the eye lens and in lens tissues, there is little information regarding intracellular protein degradation in intact lens cells in vitro. Cultured lens cells may be useful in the study of intracellular protein degradation in the lens, a tissue with a wide range of protein half-lives. This is of interest because alterations in protein turnover in the lens have been implicated in cataract formation. This study examines intracellular protein degradation in cultured bovine lens epithelial cells (BLEC). Cell cultures were incubated with radiolabeled leucine to label intracellular proteins. Protein degradation was measured by monitoring the release of trichloroacetic-acid-soluble radioactivity into the culture medium. The average half-life of long-lived proteins (half-life >50 h) was typically about 57 h in serum-supplemented medium. Average rates of degradation of long-lived proteins increased by up to 73% when fetal bovine serum was withdrawn from the culture medium. Serum had no effect on the degradation of short-lived proteins (half-life <10 h). Degradation of long-lived proteins in the presence and absence of serum was further studied in cultured BLEC from population doubling level (PDL) 2 to 43. Average half-life of proteins in serum-supplemented medium was 52 to 58 h and did not vary significantly as a function of PDL. Degradation rates in serum-free medium increased approximately twofold up to PDL 7, but returned by PDL 25 to original levels, which were maintained through PDL 43. This work was supported in part by grants from U. S. Department of Agriculture contract 53-3K06-5-10, Massachusetts Lions Eye Research Fund, Inc., and the Daniel and Florence Guggenheim Foundation. D. A. E. is a recipient of a National Eye Institute postdoctoral fellowship.     

Growth regulation of the mammalian ocular lens by vitreous humor.

Experiments were performed in our laboratory to study the effects of a mammalian 8 kD vitreous humor (VH) factor on the DNA synthesis and mitosis of the epithelial cells of organ cultured rabbit lens. The 8 kD polypeptide factor was purified from mature rabbit vitreous humor by liquid chromatography. Proliferative activities of the epithelial cells of organ cultured lenses were stimulated by 3% rabbit serum. The data from our experiments depicted that the 8 kD VH factor effectively inhibits DNA synthesis and mitosis by the epithelial cells of the organ cultured lens. Our experiments also showed that this 8 kD VH factor can maintain its growth inhibitory activity even when heated for 3 min at 95 degrees C. The growth inhibitory effect of the 8 kD VH factor was dose dependent. Using iodinated vitreal proteins it was demonstrated that the VH proteins are able to enter or bind to lens epithelial cells. The growth inhibitory effect of the 8 kD VH factor was also tested on tissue cultured lens epithelial cells. These experiments showed that the 8 kD VH factor has no growth inhibitory effect on the tissue cultured lens epithelial cells. This experiment has been repeated many times using different concentrations of the factor. These observations suggest that the 8 kD VH factor may have receptors in the lens capsular material (extracellular matrix) and the factor-receptor binding is essential for the growth inhibitory effect.     

The eye lens has an active ubiquitin-protein conjugation system

Using exogenous 125I-ubiquitin, ubiquitin-lens protein conjugation was observed with supernatants of cultured rabbit lens epithelial cells and lens cortex tissue. Conjugation was ATP-dependent with the greatest variety and amount of conjugates larger than 150 kDa. In vivo production of ubiquitin-protein conjugates in cultured rabbit and beef lens epithelial cells and rabbit lens tissues of different developmental age was established using immunological detection. There were limited similarities between conjugates found in youngest as opposed to oldest tissue. Cultured rabbit cells contained 27 pmol/mg free ubiquitin and 18 pmol/mg conjugated ubiquitin. Levels of free ubiquitin in lens tissue epithelium, cortex, and core were 36, 5, and 5 pmol/mg, respectively. There were only 2 pmol/mg conjugated ubiquitin in each of these tissues. Hydrolysis of 125I-ubiquitin was catalyzed by supernatants of cultured lens cells, beef and human lens tissues, and reticulocytes. Degradation was greatest in epithelial tissues, and least in core. This corroborates studies which show that proteolytic capabilities are attenuated in older tissue. Decreased initiation of proteolysis by ubiquitination as well as diminished proteolysis in older lens tissue may be related to the accumulation of damaged proteins in aging lens tissue.     

Heat shock proteins of adult and embryonic human ocular lenses.

We investigated the presence and distribution of heat shock proteins, HSP-70 [Horwitz, J. 1992. Proc Natl Acad Sci 89:10449-10453], HSP-40, HSc-70, HSP-27, and alphabeta-crystallin in different regions of adult and fetal human lenses and in aging human lens epithelial cells. This study was undertaken because heat shock proteins may play an important role in the maintenance of the supramolecular organization of the lens proteins. Human adult and fetal lenses were dissected to separate the epithelium, superficial cortex, intermediate cortex, and nucleus. The water soluble and insoluble protein fractions were separated by SDS-PAGE, and transferred to nitrocellulose paper. Specific antibodies were used to identify the presence of heat shock proteins in distinct regions of the lens. HSP-70 [Horwitz, 1992], HSP-40, and HSc-70 immunoreactivity was mainly detected in the epithelium and superficial cortical fiber cells of the adult human lens. The small heat shock proteins, HSP-27 and alphabeta-crystallin were found in all regions of the lens. Fetal human lenses showed immunoreactivity to all heat shock proteins. An aging study revealed a decrease in heat shock protein levels, except for HSP-27. The presence of HSP-70 [Horwitz, 1992], HSP-40, and HSc-70 in the epithelium and superficial cortical fiber cells imply a regional cell specific function, whereas the decrease of heat shock protein with age could be responsible for the loss of optimal protein organization, and the eventual appearance of age-related cataract.     

The Ocular Lens Epithelium

An adult lens contains two easily discernible, morphologically distinct compartments, the epithelium and the fiber-cell mass. The fiber-cell mass provides the lens with its functional phenotype, transparency. Metabolically, in comparison to the fiber cells the epithelium is the more active compartment of the ocular lens. For the purposes of this review we will only discuss the surface epithelium that covers the anterior face of the adult ocular lens. This single layer of cells, in addition to acting as a metabolic engine that sustains the physiological health of this tissue, also works as a source of stem cells, providing precursor cells, which through molecular and morphological differentiation give rise to fiber cells. Morphological simplicity, defined developmental history and easy access to the experimenter make this epithelium a choice starting material for investigations that seek to address universal questions of cell growth, development, epithelial function, cancer and aging. There are two important aspects of the lens epithelium that make it highly relevant to the modern biologist. Firstly, there are no known clinically recognizable cancers of the ocular lens. Considering that most of the known malignancies are epithelial in origin this observation is more than an academic curiosity. The lack of vasculature in the lens may explain the absence of tumors in this tissue, but this provides only a teleological basis to a very important question for which the answers must reside in the molecular make-up and physiology of the lens epithelial cells. Secondly, lens epithelium as a morphological entity in the human lens is first recognizable in the 5th–6th week of gestation. It stays in this morphological state as the anterior epithelium of the lens for the rest of the life, making it an attractive paradigm for the study of the effects of aging on epithelial function. What follows is a brief overview of the present status and lacunae in our understanding of the biology of the lens epithelium.     

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.     

Upregulation of Glyoxalase I fails to Normalize Methylglyoxal Levels: A Possible Mechanism for Biochemical Changes in Diabetic Mouse Lenses

Glyoxalase I is the first enzyme in a two-enzyme glyoxalase system that metabolizes physiological methylglyoxal (MGO). MGO reacts with proteins to form irreversible adducts that may lead to crosslinking and aggregation of lens proteins in diabetes. This study examined the effect of hyperglycemia on glyoxalase I activity and its mRNA content in mouse lens epithelial cells (mLE cells) and in diabetic mouse lenses and investigated the relationship between GSH and MGO in organ cultured lenses. mLE cells cultured with 25 mM D-glucose (high glucose) showed an upregulation of glyoxalase I activity and a higher content of glyoxalase I mRNA when compared with either cells cultured with 5 mM glucose (control) or with 20 mM L-glucose + 5 mM D-glucose. MGO concentration was significantly elevated in cells cultured with high D-glucose, but not in L-glucose. GSH levels were lower in cells incubated with high glucose compared to control cells. Glyoxalase I activity and mRNA levels were elevated in diabetic lenses compared to non-diabetic control mouse lenses. MGO levels in diabetic lenses were higher than in control lenses. Incubation of lenses with buthionine sulfoximine (BSO) resulted in a dramatic decline in GSH but the MGO levels were similar to lenses incubated without BSO. Our data suggest that in mouse lenses MGO accumulation may occur independent of GSH concentration and in diabetes there is an upregulation of glyoxalase I, but this upregulation is inadequate to normalize MGO levels, which could lead to MGO retention and chemical modification of proteins.     

人多潜能胚胎生殖细胞的分离和培养

多潜能胚胎性干细胞来源有两条途经,从植入前的早期胚胎内细胞团(inner cell mass,ICM)分离出来的称胚胎干细胞(embryonic stem cells,ES);从原始生殖细胞(primordial germ cells,PGCs)分离得到的称胚胎生殖细胞(embryonic germ cells,EG)。这两种干细胞在小鼠嵌合体实验中,都证明具有参与生殖系传递的能力。这类干细胞在体外保持     

Increased expression of nonmuscle myosin IIs is associated with 3MC-induced mouse tumor

Administration of the chemical carcinogen, 3-methylcholanthrene (3MC), in the hind leg induces the progressive formation of tumors in mice within 110 days. Previous reports suggest that transformation of muscle cells to atypical cells is one of the causes of tumor formation. Molecular events that lead to transformation of normal cells to atypical cells are not well understood. Here, we investigate the effect of 3MC on the expression of nonmuscle myosin IIs (NM IIs) which are known to be involved in cell migration, division and adhesion. Mass spectroscopy analysis reveals that tumor tissue contains 64.5% NM II-A, 34% II-B and only 1.5% II-C of total NM IIs, whereas these three isoforms of NM IIs are undetectable by mass spectroscopy in normal tissue associated with the tumor (NTAT) from the hind leg. Quantification of heavy chain mRNAs of NM II suggests that tumor tissue contains 25.7-fold and 19.03-fold more of NM II-A and II-B, respectively, compared with NTAT. Unlike NM II-B, which is detected only after tumor formation, II-A is detectable as early as day 7 after a second dose of 3MC. Immunofluorescence confocal microscopy reveals that fibroblast cells which are sparsely distributed in normal tissue are densely populated but of atypical shape in the tumor. These findings suggest that transformation of fibroblasts or non-fibroblast cells to atypical, cancerous cells is associated with increased levels of NM II-A and NM II-B expression in the 3MC-induced tumor mouse model. 3MC-induced transformation is further demonstrated in C2C12 myotubes.     

人多潜能胚胎生殖细胞的分离和培养（简报）

To establish human pluripotent embryonic germ (EG) cell lines, human primordial germ cells (PGCs) of embryos aborted in 5-9 week were cultured on inactive mouse STO fibroblast feeder. The medium contained human leukemia inhibitory factor (hLIF), human basic fibroblast growth factor (hbFGF) and forskolin. The EG cells could be passaged continuously until 12 generations. Most cells were positive in alkaline phosphatase staining and expressed cell surface antigen SSEA-3 and pluripotent marker Oct-4. These EG cell populations that retained normal karyotype could form embryoid body in culture and differentiate further into neuron-like cells, mucous epithelial cells, epithelial cells and other types of the cells spontaneously. These results indicated the cell clones derived from human PGCs resemble pluripotent EG cells from mouse PGCs in appearance or nature.     

Differential timing of nuclear lamin A/C expression in the various organs of the mouse embryo and the young animal: a developmental study

In mouse embryos, acquisition of the nuclear lamin polypeptides A/C varies according to developmental stage and tissue type. In order to determine the precise time points and cell types in which lamin A/C are first observed, we have used two monoclonal antibodies in immunofluorescence studies of different tissues of developing mouse embryos and of young mice. One antibody (mAB346) is specific for lamins A and C, while the other (PKB8) detects lamins A, B and C. Dividing uterine development into three phases--germ layer formation, organogenesis and tissue differentiation--our results show that lamin A/C expression in the embryo proper is not observed until the third phase of development. Lamin A/C first appears at embryonic day 12 in muscle cells of the trunk, head and the appendages. Three days later it is also seen in cells of the epidermis where its appearance coincides with the time of stratification. In the simple epithelial of lung, liver, kidney and intestine, as well as in heart and brain, lamins A/C do not appear until well after birth. Embryonal carcinoma (EC) cells express lamin B but not lamin A/C. Lamin A/C expression is noted in some EC cells after they are induced to differentiate and in several differentiated teratocarcinoma cell lines. Our results suggest that commitment of a cell to a particular pathway of differentiation (assayed by cell-type-specific expression of intermediate filament proteins) usually occurs prior to the time that lamin A/C can be detected. Thus lamin A/C expression may serve as a limit on the plasticity of cells for further developmental events.     

Anchorage-independent culture maintains prostate stem cells

Freshly isolated mouse prostate epithelial cells regenerate fully differentiated prostate tissue when combined with embryonic urogenital sinus mesenchyme and grafted in vivo. We show here that this regenerative capacity, which has been attributed to a small population of pleuripotential progenitor epithelial cells, is rapidly lost when the cells are placed in monolayer culture but can be maintained by culture in anchorage-independent conditions. Epithelial cells placed in anchorage-independent culture formed proliferating spheres that could be serially passaged and exhibited increased expression of putative stem cell markers as compared to cells grown in monolayer culture. Epithelial cells isolated from the fetal urogenital sinus, the newborn, and adult prostate formed spheres with similar efficiency, while cells isolated from the post-castration prostate exhibited significantly higher sphere-forming abilities. When passaged spheres were recombined with E17 rat urogenital sinus mesenchyme and grafted in vivo, they generated fully differentiated mouse prostate glandular epithelium containing both p63+ basal cells and p63− luminal cells and expressing a variety of prostate-specific and terminal differentiation markers.     

Developmentally regulated expression of hemoglobin subunits in avascular tissues

We investigated the spatio-temporal profile of hemoglobin subunit expression in developing avascular tissues. Significant up-regulation of hemoglobin subunits was identified in microarray experiments comparing blastocyst inner cell masses with undifferentiated embryonic stem (ES) cells. Hemoglobin expression changes were confirmed using embryoid bodies (derived from in vitro differentiation of ES cells) to model very early development at pre-vascular stages of embryogenesis; i.e. prior to hematopoiesis. We also demonstrate, using RT-PCR, Western blotting and immunocytochemistry, expression of adult and fetal mouse hemoglobin subunits in the avascular ocular lens at various stages of development and maturation. Hemoglobin proteins were expressed in lens epithelial cells (cytoplasmic) and cortical lens fiber cells (nuclear and cell-surface-associated); however, a sensitive heme assay demonstrated negligible levels of heme in the developing lens postnatally. Hemoglobin expression was also observed in the developing eye in corneal endothelium and retinal ganglion cells. Gut sections showed, in addition to erythrocytes, hemoglobin protein staining in rare, individual villus epithelial cells. These results suggest a paradigm shift: hemoglobin subunits are expressed in the avascular lens and cornea and in pre-hematopoietic embryos. It is likely, therefore, that hemoglobin subunits have novel developmental roles; the absence of the heme group from the lens would indicate that at least some of these functions may be independent of oxygen metabolism. The pattern of expression of hemoglobin subunits in the perinuclear region during lens fiber cell differentiation, when denucleation is taking place, may indicate involvement in the apoptosis-like signaling processes occurring in differentiating lens fiber cells.     

Aging and cellular maturation cause changes in ubiquitin-eye lens protein conjugates

The eye lens is a useful tissue for studying phenomena related to aging since it can be separated into differentially aged or matured zones. This work establishes correlations between ubiquitin-lens protein conjugating capabilities and age, as well as the stage of maturation of bovine lens tissue. When exogenous 125I-ubiquitin was combined with supernatants of epithelial (least mature), cortex, and core (most mature) tissue, ATP-dependent conjugation of 125I-ubiquitin to lens proteins was most effective with the epithelial tissue preparation. Conjugate formation was greatest when lenses were obtained from young animals. Supernatants from cultured bovine lens epithelial (BLE) cells conjugated more 125I-ubiquitin to lens proteins than any tissue preparation. In all cases the predominant conjugates formed in these cell-free assays were of high molecular mass, although conjugates with masses in the 25-70 kDa range were also observed. Lens tissue and cultured BLE cell preparations were also probed with antibodies to ubiquitin to detect in vivo ubiquitin-lens protein conjugates. There was more free ubiquitin and ubiquitin conjugates in tissue from young as compared with older lenses. The greatest levels of conjugates were observed in cultured BLE cells. Specificity in the ubiquitination system is indicated since some of the conjugates formed in vivo appear identical to those formed in the cell-free assays and in reticulocytes using exogenous 125I-ubiquitin. Upon development and maturation of lens tissue (i.e., core as opposed to epithelium), there is accumulation of lower molecular mass conjugates.     

AGEs in human lens capsule promote the TGFβ2‐mediated EMT of lens epithelial cells: implications for age‐associated fibrosis

Proteins in basement membrane (BM) are long‐lived and accumulate chemical modifications during aging; advanced glycation endproduct (AGE) formation is one such modification. The human lens capsule is a BM secreted by lens epithelial cells. In this study, we have investigated the effect of aging and cataracts on the AGE levels in the human lens capsule and determined their role in the epithelial‐to‐mesenchymal transition (EMT) of lens epithelial cells. EMT occurs during posterior capsule opacification (PCO), also known as secondary cataract formation. We found age‐dependent increases in several AGEs and significantly higher levels in cataractous lens capsules than in normal lens capsules measured by LC‐MS/MS. The TGFβ2‐mediated upregulation of the mRNA levels (by qPCR) of EMT‐associated proteins was significantly enhanced in cells cultured on AGE‐modified BM and human lens capsule compared with those on unmodified proteins. Such responses were also observed for TGFβ1. In the human capsular bag model of PCO, the AGE content of the capsule proteins was correlated with the synthesis of TGFβ2‐mediated α‐smooth muscle actin (αSMA). Taken together, our data imply that AGEs in the lens capsule promote the TGFβ2‐mediated fibrosis of lens epithelial cells during PCO and suggest that AGEs in BMs could have a broader role in aging and diabetes‐associated fibrosis.