The tyrosine phosphorylation substrate p36 is developmentally regulated in embryonic avian limb and is induced in cell culture

The 36-kD protein-tyrosine kinase substrate p36 has been variously postulated to be involved in membrane-cytoskeletal interactions, membrane traffic, and the regulation of phospholipase A2, and its phosphorylation may play some role in malignant transformation by avian sarcoma viruses. Because embryonic tissues are resistant to transformation by avian sarcoma viruses, we have examined the expression of p36 in the developing avian embryonic limb. The level of p36 increased progressively from day 5 to day 14 of development. It was largely absent from day-5 mesenchyme, and was induced during the differentiation of mesenchymal cells into connective tissue and cartilage, but was not induced in differentiating muscle. In contrast, p36 was detected in ectodermal cells at all developmental stages examined. When day-5 limbs were dissociated and cultured, p36 was induced in all adherent cells, beginning at 2-4 h after plating, and reaching levels comparable to those observed with intact day-14 limb tissue within 48 h. The accumulation of p36 in culture was dependent on substratum adherence, suggesting that its stability is regulated by cell attachment or spreading. These findings are consistent with a structural or mechanical role for p36.     

Changes in the distribution of the 34-kdalton tyrosine kinase substrate during differentiation and maturation of chicken tissues

We examined the distribution of the 34-kilodalton (34-kD) tyrosine kinase substrate in tissues of adult and embryonic chicken using both a mouse monoclonal antibody and a rabbit polyclonal antibody raised against the affinity purified 34 kD protein. We analyzed the localization by immunoblotting of tissue extracts, by immunofluorescence staining of frozen tissue sections, and by staining sections of paraffin-embedded organs by the peroxidase antiperoxidase method. The 34-kD protein was present in a variety of cells, including epithelial cells of the skin, gastrointestinal, and respiratory tracts, as well as in fibroblasts and chondrocytes of connective tissue and mature cartilage, and endothelial cells of blood vessels. The 34-kD protein was also found in subpopulations of cells in thymus, spleen, bone marrow, and bursa. The protein was not detected in cardiac, skeletal, or smooth muscle cells, nor in epithelial cells of liver, kidney, pancreas, and several other glands. Although most neuronal cells did not contain the 34-kD protein, some localized brain regions did contain detectable amounts of this protein. The 34-kD protein was not detected in actively dividing cells of a number of tissues. Changes in the distribution of the 34-kD protein were observed during the differentiation or maturation of cells in several tissues including epithelial cells of the skin and gastrointestinal tract, fibroblasts of connective tissue, and chondroblasts.     

Tissue distribution and immunohistochemical localization of the collagen-binding heat-shock protein gp46 in neonatal rats

Summary Collagen-binding heat-shock proteins ofM _r 46–47 kDa have been postulated to function as putative molecular chaperones in the biosynthesis of collagen in several species. The rat homologue of this family of heat-shock proteins is called gp46. In the present study, we employed Western blotting and immunohistochemical methods to determine the tissue distribution and cellular localization of gp46 in the thoracic aorta, heart, kidney, liver and lung of eight-day-old Wistar rats. Highest levels of gp46 were detected in the thoracic aorta and lung, followed by the kidney and heart. Gp46 levels were low to undetectable by Western blot analysis in the liver. Immunohistochemistry revealed that gp46 labelling was observed almost exclusively in three distinct cell types: fibroblasts, muscle cells, and some epithelial cells. Gp46 was detected in the fibroblasts of the hepatic triad, in the interstitium of the alveolar wall and in the tunica adventitia of blood vessels in the majority of tissues examined, in atrial and ventricular cardiomyocytes, in vascular smooth muscle cells of the abluminal portion of the tunica media, in parietal epithelial cells and mesangial cells of the glomerulus, in epithelial cells of the distal tubules and collecting ducts in the kidney and clusters of immature renal tubules, in epithelial cells of the bile duct, and in mesodermal cells surrounding the liver. These results demonstrate that gp46 is present in collagen producing cells and cells undergoing rapid growth and development, suggesting that gp46 may play a significant role in these processes.     

Ataxia-telangiectasia mutated is not required for p53 induction and apoptosis in irradiated epithelial tissues

The ataxia-telangiectasia mutated (Atm) protein kinase is a central regulator of the cellular response to DNA damage. Although Atm can regulate p53, it is not known if this Atm function varies between tissues. Previous studies showed that the induction of p53 and apoptosis by whole-body ionizing radiation varies greatly between tissue and tumor types, so here we asked if Atm also had a tissue-specific role in the ionizing radiation response. Irradiated Atm-null mice showed impaired p53 induction and apoptosis in thymus, spleen, and brain. In contrast, radiation-induced p53, apoptosis, phosphorylation of Chk2, and G(2)-M cell cycle arrest were slightly delayed in Atm(-/-) epithelial cells of the small intestine but reached wild-type levels by 4 h. Radiation-induced p53 and apoptosis in Atm(-/-) hair follicle epithelial cells were not impaired at any of the time points examined. Thus, Atm is essential for radiation-induced apoptosis in lymphoid tissues but is largely dispensable in epithelial cells. This indicates that marked differences in DNA damage signaling pathways exist between tissues, which could explain some of the tissue-specific phenotypes, especially tumor suppression, associated with Atm deficiency.     

Tyrosine phosphorylation of a 22-kDa protein is correlated with transformation by Rous sarcoma virus

Recent studies from this laboratory have identified novel cytoskeletal proteins that are phosphorylated on tyrosine in vivo in Rous sarcoma virus-transformed chick fibroblasts (Glenney, J. R., Jr., and Zokas, L. (1989) J. Cell Biol. 108, 2401-2408). In the present report, the phosphorylation of these proteins was examined in cells expressing the nonmyristylated mutants of src that are not transformed. A good correlation was found between transformation and the tyrosine phosphorylation of a 22-kDa protein. Tyrosine phosphorylation of the 22-kDa protein was reduced more than 95% in cells expressing the nonmyristylated mutants of src. Size fractionation revealed that the 22-kDa phosphoprotein in transformed chick fibroblasts is found in a Mr 150,000 complex. Monoclonal antibodies were used to screen various chicken tissues where the 22-kDa protein was found at high levels in muscle and lung with low levels in epithelial cells and brain. The 22-kDa protein becomes an excellent candidate for a mediator of transformation by the tyrosine kinase class of oncogenes.     

Biochemical and histochemical characteristics of proteins homologous to calf lens membrane proteins with high calcium-binding capacity

Proteins with high affinity and capacity for calcium are present in the membranes of calf lens fiber and epithelial cells. They can be extracted from these membranes by means of EDTA or EGTA. The tissue specificity and localization of these 30-38 kD EDTA-extractable proteins (EEP) have been examined. Antibodies raised against calf lens fiber EEP specifically form immune complexes with distinct proteins of 30-38 kD in a great variety of non-lenticular tissues. By indirect immunofluorescence microscopy using anti-EEP antiserum, the EEP-like proteins could be detected in fibroblasts, retinal Müller cells, endothelial cells and some types of epithelial cells. Only covering epithelia (cornea, glomerulus) contained significant amounts of these proteins, irrespective of the shape of the cells. EEP-like proteins were absent in secreting epithelial cells of liver, kidney tubules and pancreas. In addition, they were not detected in muscle, nerve and fat cells, erythrocytes and lymphocytes. The localization and the number of EEP-like proteins varied among different cell types. In fibroblasts, containing only two EEP-like proteins (molecular weight (MW) 33.0 and 31.5 kD in calf tissue), predominantly the nucleus was stained. In vitro studies with permeabilized cultured fibroblasts from several species have shown that the nuclear staining was built up of bright spots around unstained nucleoli. In epithelial and endothelial cells of calf tissue, however, most fluorescent label was found in the plasma membranes. Immunoblotting experiments revealed the presence in these cell types of at least five EEP-like proteins, including a 33.0 and 31.5 kD component. The difference in staining pattern between these cells and fibroblasts might thus indicate that the nature or the localization of some of the EEP-like proteins is cell type-specific. Because of their extractability from various tissue membrane fractions by means of EDTA or EGTA it is suggested that at least part of the EEP-like proteins is bound to membrane structures via calcium. This characteristic feature, together with the MW values and the cross-reactivity with anti-EEP antiserum indicate that these proteins and the lens membrane proteins with high calcium-binding capacity share a very high degree of homology and may even be identical.     

Lipoprotein lipase stored in adipocytes and muscle cells is a cryptic enzyme

The status of lipoprotein lipase (LPL) has been examined in different cell types (adipose, skeletal muscle, and heart muscle cells) and different tissues (adipose, muscle, and cardiac tissues) from mouse, rat, and human. Cell and secreted activities were compared in cycloheximide-, heparin-treated cells present in culture. A gross underestimation of cell LPL activity was found; excess of LPL over substrate and/or apolipoprotein C-II was excluded as well as inhibition by cell component(s) or detergent molecules used to disrupt membrane structures in the cell lysates. Unmasking of LPL activity occurred upon dilution: the higher the concentration of LPL, the higher were the dilution factor and the concentration of heparin required to reach a plateau of activity. This maximal value was found to be identical to that determined in the secretion medium, indicating that the cell LPL activity can be determined in toto. The unmasking effect of dilution upon LPL activity was extended to adipose, muscle, and cardiac tissues from rat and to adipose tissues from mouse and human. In agreement with previous results (Vannier et al., 1989, J. Biol. 264: 13199-13205), our results are in favor of LPL as being cryptic within the cell. A model is proposed, in which potentially active LPL molecules are present as aggregates in various membrane compartments. It is concluded that the determination of the pool size of catalytically active cell LPL has to be estimated in vitro under the appropriate conditions described herein.     

Expression of mouse Fbxw7 isoforms is regulated in a cell cycle- or p53-dependent manner

Fbxw7 is the F-box protein component of an SCF-type ubiquitin ligase that contributes to the ubiquitin-dependent degradation of cell cycle activators and oncoproteins. Three isoforms (alpha, beta, and gamma) of Fbxw7 are produced from mRNAs with distinct 5' exons. We have now investigated regulation of Fbxw7 expression in mouse tissues. Fbxw7alpha mRNA was present in all tissues examined, whereas Fbxw7beta mRNA was detected only in brain and testis, and Fbxw7gamma mRNA in heart and skeletal muscle. The amount of Fbxw7alpha mRNA was high during quiescence (G0 phase) in mouse embryonic fibroblasts (MEFs) and T cells, but it decreased markedly as these cells entered the cell cycle. The abundance of Fbxw7alpha mRNA was unaffected by cell irradiation or p53 status. In contrast, X-irradiation increased the amount of Fbxw7beta mRNA in wild-type MEFs but not in those from p53-deficient mice, suggesting that radiation-induced up-regulation of p53 leads to production of Fbxw7beta mRNA. Our results thus indicate that expression of Fbxw7 isoforms is differentially regulated in a cell cycle- or p53-dependent manner.     

A role for SSeCKS, a major protein kinase C substrate with tumour suppressor activity, in cytoskeletal architecture, formation of migratory processes, and cell migration during embryogenesis

SSeCKS is a major protein kinase C substrate which has tumour suppressor activity in models of src- and ras-induced oncogenic transformation. The mitogenic regulatory activity of SSeCKS is likely manifested by its ability to bind key signalling proteins such as protein kinases C and A and calmodulin, and to control actin-based cytoskeletal architecture. Rat SSeCKS shares extensive homology with human Gravin, an autoantigen in myasthenia gravis that encodes kinase scaffolding functions and whose expression pattern in fibroblasts and nerves suggests a role in cell motility. Here, we analyse the expression of SSeCKS and Gravin in rodent and human fibroblast and epithelial cell lines using antibodies specific or crossreactive for SSeCKS or Gravin. SSeCKS expression was then analysed in developing mouse embryos and in adult tissues. In the foetal mouse, early SSeCKS protein expression (E10–11) is focused in the loose mesenchyme, luminal surface of the neural tube, notochord, early heart and pericardium, urogenital ridge, and dorsal and ventral sections of limb buds. In later stages (E12–14), SSeCKS is widely expressed in mesenchymal cells but is absent in the spinal ganglia. By E15, SSeCKS expression is ubiquitous, although the staining pattern varies from being striated within smooth muscle sarcomeres to filamentous in mesenchymal and select epithelial cells. In the adult mouse, SSeCKS staining is relatively ubiquitous, with highest expression in the gonads, smooth and cardiac muscle, lung, brain and heart. High expression is also detected in fibroblasts and nerve fibres as well as in more specialized cells such as glomerular mesangial cells and testicular Sertoli cells. SSeCKS expression in the rat testes correlates with the induction of puberty, and in mature mouse spermatozoa, SSeCKS is found in peripheral acrosome membranes and in a helix-like winding pattern within the midsection. Periodic enrichments of SSeCKS are found in sperm midsections and in developing axons, suggesting a role in architectural infrastructure. As with Gravin, high SSeCKS expression is absent in most epithelial cells; however, in contrast to Gravin, SSeCKS is expressed in Purkinje cells, cardiac muscle, macrophages and hepatic stellate cells, indicating overlapping yet distinct patterns of tissue expression in the SSeCKS/Gravin family. The data suggest roles for SSeCKS in the control of cytoskeletal and tissue architecture, formation of migratory processes and cell migration during embryogenesis.     

Distribution of peptidases in cultured cells from middle ear mucosa: prolyl endopeptidase is localized in fibroblasts and dipeptidyl peptidase IV and II in epithelial cells.

To examine the distribution of prolyl endopeptidase (PEP), dipeptidyl peptidase IV (DPP IV), and dipeptidyl peptidase II (DPP II) in specific cell types, fibroblasts and epithelial cells were selectively cultured from middle ear mucosal tissues of guinea pigs. In fibroblasts, PEP had the highest activity, 12.28 +/- 4.00 nmole/min/mg protein (mean +/- SD), 45-fold higher than corresponding DPP II levels. In epithelial cells, DPP IV activity was the highest, 6.48 +/- 0.90 nmole/min/mg protein. This communication describes, for the first time, the distribution of the enzyme activities of PEP, DPP IV, and DPP II in fibroblasts and epithelial cells, and the occurrence of PEP in fibroblasts.     

Roles of the Ras-MEK-mitogen-activated protein kinase and phosphatidylinositol 3-kinase-Akt-mTOR pathways in Jaagsiekte sheep retrovirus-induced transformation of rodent fibroblast and epithelial cell lines

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA), a transmissible lung cancer of sheep. The virus can induce tumors rapidly, and we previously found that the JSRV envelope protein (Env) functions as an oncogene, because it can transform mammalian and avian fibroblast cell lines. (N. Maeda, Proc. Natl. Acad. Sci. USA 98:4449-4454, 2001). The molecular mechanisms of JSRV Env transformation are of considerable interest. Several reports suggested that the phosphatidylinositol 3-kinase/Akt pathway is important for transformation of mammalian fibroblasts but not for chicken fibroblasts. In this study, we found that Akt/mTOR is involved in JSRV transformation of mouse NIH 3T3 fibroblasts, because treatment with the mTOR inhibitor rapamycin reduced transformation. We also found that H/N-Ras inhibitor FTI-277 and MEK1/2 inhibitors PD98059 and U0126 strongly inhibited JSRV transformation of NIH 3T3 fibroblasts, suggesting that the H/N-Ras-MEK-mitogen-activated protein kinase (MAPK) p44/42 pathway is necessary for the transformation. In RK3E epithelial cells, the MEK1/2 inhibitors also eliminated transformation, but FTI-277 only partially inhibited transformation. It was noteworthy that p38 MAPK inhibitors enhanced JSRV transformation in both fibroblasts and epithelial cells. Treatment of transformed cells with p38 inhibitors both increased levels of phospho-MEK1/2 and phospho-p44/42 and induced rapid enhancement of the transformed phenotype. Immunohistochemical staining of tumor tissues from naturally and experimentally induced OPA and naturally occurring enzootic nasal adenocarcinoma revealed strong activation of MAPK p44/42 in all cases examined. However, p38 activation was not generally observed. These results indicate that signaling through two pathways (in particular, H/N-Ras-MEK-MAPK and, to a lesser extent, Akt-mTOR) is important for JSRV-induced transformation and that p38 MAPK has a negative regulatory effect on transformation, perhaps via MEK1/2 and p44/42.     

Nonuniform behavior of multiple isoactins in the same cell is a cell-dependent phenomenon

The functional significance of multiple isoactins in the same cell is still not understood. To address this question, we examined the response of smooth muscle and cardiac muscle alpha-isoactins to a serial extraction procedure applied to both muscle and nonmuscle cell types. We compared these extraction results with results obtained with the beta- and gamma-nonmuscle actin isoforms from the same cells. In differentiated BC3H1 nonfusing muscle cells (smooth muscle alpha-isoactin), in human rhabdomyosarcoma cells (cardiac alpha-isoactin), and in chick skeletal muscle cells (cardiac alpha-isoactin), different fractions were found selectively enriched in either the nonmuscle or the muscle-specific actin isoforms compared with their relative abundance in whole cell extracts. Conversely, when these same isoactins were examined either in undifferentiated BC3H1 cells or in mouse nonmuscle cells stably transfected with a cardiac alpha-isoactin gene, no enrichment of these isoforms above their relative abundance in whole cell extracts was observed. These results indicate that within the muscle or muscle-like cells examined, the different actin isoforms were either selectively utilized or localized. These results further show that isoactin-specific responses observed were apparently related to the cell type in which they were found and not to differences in inherent physical properties such as solubility of the different isoactins examined.     

Splicing of two alternative exon pairs in beta-tropomyosin pre-mRNA is independently controlled during myogenesis.

Two known tissue-specific tropomyosin (TM) isoforms are produced from the rodent beta-TM gene. Skeletal muscle beta-TM uses the alternative exons 6b and 9a and the exon 9a-associated poly(A) site. Fibroblast and smooth muscle TM-1 use exons 6a and 9b and the exon-9b associated poly(A) site. We have identified a new skeletal muscle beta-TM isoform, beta-TM2. beta-TM2 contains exon 6b (muscle) and exon 9b (nonmuscle). Full-length beta-TM2 cDNA clones were isolated from a cDNA library of mouse muscle BC3H1 cells. Its mRNA was also found in mouse skeletal muscle tissue but not in other tissues. beta-TM2 mRNA level and protein synthesis are differentiation-dependent, with a transient high level in the early stages of myogenesis both in BC3H1 cells and in mouse embryo limbs. Trace amounts of beta-TM3 mRNA, the other hybrid form (exons 6a + 9a), were found in less differentiated BC3H1 cells, mouse uterus, heart, and 3T3 fibroblasts but not skeletal muscle tissue. Thus, the selection of the two alternative exons appears to be controlled independently. Furthermore, during myogenesis, there is a sequential switch in the internal alternative exon, the terminal exon, and the poly(A) site from the nonmuscle to the muscle type.     

Soluble age-related factors from skeletal muscle which influence muscle development

Successful regeneration of damaged striated muscle in adult mice is dependent on the regeneration of newly differentiated myofibers from proliferating satellite cells and inhibition of scar tissue formation by fibroblasts. As with most tissues, the ability of skeletal muscle to regenerate decreases in older animals. In this study, we have analysed soluble extracts from intact and regenerating skeletal muscle from mice of different ages for their ability to affect avian myogenesis in tissue culture. We were interested in determining whether an age-dependent difference could be detected with this tissue culture bioassay system. Total cell proliferation in the cultures, measured by [3H]thymidine incorporation was increased equally by muscle extracts from both young and older mice but the resulting cell populations differed in proportion of cell types. The ratio of myoblasts to fibroblasts was significantly greater in cultures exposed to extracts from younger mouse muscle as compared with cultures exposed to extracts from older animals. This age-related activity was found to reside in a low molecular weight (MW) (greater than 12 kD) component of the extract. This fraction had dissimilar effects on myoblasts and fibroblasts. Relative to saline controls, myoblast proliferation was increased and fibroblast proliferation decreased. The low MW fraction from younger mouse muscle extracts stimulated myogenic cell proliferation and myotube formation to a greater extent than the similar fraction prepared from older mouse muscle. Conversely, younger mouse muscle fractions had significantly greater inhibitory activity against fibroblast proliferation than did older mouse muscle fractions.     

Some lymphoid cell lines transformed by Abelson murine leukemia virus lack a major 36,000-dalton tyrosine protein kinase substrate.

Fibroblasts transformed by Abelson murine leukemia virus differ from normal fibroblasts in that they contain several cellular proteins, including one of 29 and one of 36 kilodaltons, which are phosphorylated at tyrosine residues. Since it has been shown before that these proteins also become phosphorylated at tyrosine after transformation of fibroblasts by a number of other retroviruses, their phosphorylation may play an important role in the transformation of these cells. In contrast, the 36-kilodalton phosphoprotein was not detectable in three of the four lines of Abelson virus-transformed B lymphoma cell lines studied here. These three cell lines, RAW307.1.1, 18-48, and 18-81, and a B lymphoma induced by mineral oil, WEHI 279, were all found to lack both the phosphorylated and unphosphorylated forms of the 36-kilodalton protein. It thus appears that expression of this major cell protein is not essential for the survival of B lymphoma cells in culture and that the phosphorylation of the 36-kilodalton protein at tyrosine is not essential for transformation of pre-B lymphocytes by Abelson virus.     

Three mouse monoclonal antibodies to human differentiation antigens: reactivity with two mucin-like antigens and with connective tissue fibers

Three human differentiation antigens (MU78, MT334, and MQ49) have been defined by mouse monoclonal antibodies developed from mice immunized with ovarian carcinoma cell lines. Their distribution was determined on 148 cultured cell lines of various histologic types and on frozen sections of 16 normal tissues. MU78 was found in fibrillar structures in soft connective tissue with a distribution resembling that of elastin fibers; however, elastin fibers in elastic cartilage and in the aorta were nonreactive. MU78 was detected in cultured carcinoma cells of various histologic types, where it had a nonfibrillar, cytoplasmic distribution, but was not detected in normal epithelial cells in frozen sections. Cultured fibroblasts, astrocytomas, melanomas, and lymphomas did not contain MU78. In cell lines, MU78 appears to be a protein of 2000-5000 daltons. The other two antigens, MT334 and MQ49, are both mucin-like molecules, and the determinants are probably carbohydrate in nature. Of the normal tissues examined, MT334 was detected only in goblet cells of the colon, though it was present in a variety of carcinomas in culture. It was detected as both a cytoplasmic and secreted component. MQ49 was detected in various secretory epithelial cells, in Hassall's corpuscles in the thymus, and in cultured carcinomas of various histologic types. It was found on the cell surface as well as in the cytoplasm and is present on a glycolipid as well as on a sulfated mucin. These results, and results of other recent studies, demonstrate the importance of mucin-like molecules as antigens in epithelial cells and secretions.     

Heregulin-alpha and heregulin-beta expression is linked to a COX-2-PGE2 pathway in human gastric fibroblasts

We have previously shown heregulin (HRG)-alpha expression in human gastric fibroblasts and its stimulation of gastric epithelial cell growth. Although cyclooxygenase (COX)-2 has also been shown to stimulate growth factor production in these cells, the interaction between COX-2 and HRG remains unknown. Conditioned media (CM) from gastric fibroblasts incubated with PGE(2) or interleukin (IL)-1beta, a well known COX-2 inducer, were analyzed for their effect on erbB3 tyrosine phosphorylation in MKN28 gastric epithelial cells. HRG protein expression in fibroblast lysates and CM was also examined by western blot. HRG-alpha and HRG-beta mRNA expression in gastric fibroblasts and human gastric tissue was examined by real-time quantitative PCR. HRG and COX-2 expressions in surgical resections of human gastric ulcer tissue were examined immunohistochemically. CM from fibroblasts incubated with PGE(2), or IL-1beta, stimulated erbB3 phosphorylation in MKN28 cells. Preincubation of the fibroblasts with celecoxib, a selective COX-2 inhibitor, suppressed CM-induced erbB3 phosphorylation. This inhibition was reversed by exogenous PGE(2). As with erbB3 phophorylation, IL-1beta stimulated both HRG-alpha and HRG-beta mRNA expression, as well as HRG release into gastric fibroblast CM. IL-1beta-stimulated HRG expression and release were also inhibited by celecoxib, and exogenous PGE(2) restored this inhibitory effect, suggesting the activation of an IL-1beta-COX-2-PGE(2) pathway that culminates in the release of HRG from fibroblasts. HRG-alpha and HRG-beta mRNA levels were significantly higher in gastric ulcer tissue than in normal gastric mucosa. HRG immunoreactivity was found in interstitial cells of the gastric ulcer bed and coexpressed with COX-2. These results suggest that HRG might be a new member of the growth factor family involved in the COX-2-dependent ulcer repair process.     

58-kDa microspherule protein (MSP58) is novel Brahma-related gene 1 (BRG1)-associated protein that modulates p53/p21 senescence pathway

The nucleolar 58-kDa microspherule protein (MSP58) protein is a candidate oncogene implicated in modulating cellular proliferation and malignant transformation. In this study, we show that knocking down MSP58 expression caused aneuploidy and led to apoptosis, whereas ectopic expression of MSP58 regulated cell proliferation in a context-dependent manner. Specifically, ectopic expression of MSP58 in normal human IMR90 and Hs68 diploid fibroblasts, the H184B5F5/M10 mammary epithelial cell line, HT1080 fibrosarcoma cells, primary mouse embryonic fibroblasts, and immortalized NIH3T3 fibroblasts resulted in induction of premature senescence, an enlarged and flattened cellular morphology, and increased senescence-associated β-galactosidase activity. MSP58-driven senescence was strictly dependent on the presence of functional p53 as revealed by the fact that normal cells with p53 knockdown by specific shRNA or cells with a mutated or functionally impaired p53 pathway were effective in bypassing MSP58-induced senescence. At least two senescence mechanisms are induced by MSP58. First, MSP58 activates the DNA damage response and p53/p21 signaling pathways. Second, MSP58, p53, and the SWI/SNF chromatin-remodeling subunit Brahma-related gene 1 (BRG1) form a ternary complex on the p21 promoter and collaborate to activate p21. Additionally, MSP58 protein levels increased in cells undergoing replicative senescence and stress-induced senescence. Notably, the results of analyzing expression levels of MSP58 between tumors and matched normal tissues showed significant changes (both up- and down-regulation) in its expression in various types of tumors. Our findings highlight new aspects of MSP58 in modulating cellular senescence and suggest that MSP58 has both oncogenic and tumor-suppressive properties.     

MAP 4: a microtubule-associated protein specific for a subset of tissue microtubules

The cytological distribution of microtubule-associated protein 4 (MAP 4) (L. M. Parysek, C. F. Asnes, J. B. Olmsted, 1984, J. Cell Biol., 99:1309-1315) in mouse tissues has been examined. Adjacent 0.5-0.9- micron sections of polyethylene glycol-embedded tissues were incubated with affinity-purified MAP 4 or tubulin antibodies, and the immunofluorescent images were compared. Tubulin antibody labeling showed distinct microtubules in all tissues examined. MAP 4 antibody also labeled microtubule-like patterns, but the extent of MAP 4 reactivity was cell type-specific within each tissue. MAP 4 antibody labeled microtubules in vascular elements of all tissues and in other cells considered to have supportive functions, including Sertoli cells in the testis and glial elements in the nervous system. Microtubule patterns were also observed in cardiac, smooth, and skeletal (eye) muscle, podocytes in kidney, Kuppfer cells in liver, and spermatid manchettes. The only MAP 4-positive cells in which the pattern was not microtubule-like were the principal cells of the collecting ducts in kidney cortex, in which diffuse fluorescence was seen. MAP 4 antibody did not react with microtubule-rich neuronal elements of the central and peripheral nervous system, skeletal muscle from anterior thigh, liver parenchymal cells, columnar epithelial cells of the small intestine, and absorptive cells of the tubular component of the nephron. These observations indicate that MAP 4 may be associated with only certain kinds of cell functions as demonstrated by the preferential distribution with microtubules of defined cell types.     

Two cholesterol ester hydrolases. Distribution in rat tissues and in cultured human fibroblasts and monkey arterial smooth muscle cells.

Hydrolytic activity against acetone-dispersed [4-14C]cholesterol oleate has been assayed as a function of pH in seven parenchymal tissues, blood cells, and plasma of the rat, as well as in cultured human fibroblasts and monkey (Macaca nemestrina) arterial smooth muscle cells. Both acid and neutral hydrolytic activities were present in all of these except rat plasma. The pH optima were in all cases close to pH 4.5 and pH 6.8. Acid activity was quite constant from tissue to tissue, while neutral activity varied greatly, being greatest in adrenal, testis, and adipose tissue. Subcellular fractionation of human fibroblasts allowed demonstration that activities at pH 4.5 and pH 6.8 were concentrated in different fractions, apparently lysosomal and polysomal, respectively. It appears most cell types, including fibroblasts and smooth muscle cells, contain two separate enzymes capable of hydrolyzing cholesterol esters. The neutral pH polysomal enzyme, which is especially prominent in certain tissues, may have a function related to the specialized roles of these tissues.