BAG-1 enhances cell-cell adhesion, reduces proliferation and induces chaperone-independent suppression of hepatocyte growth factor-induced epidermal keratinocyte migration.

Cell motility is important in maintaining tissue homeostasis, facilitating epithelial wound repair and in tumour formation and progression. The aim of this study was to determine whether BAG-1 isoforms regulate epidermal cell migration in in vitro models of wound healing. In the human epidermal cell line HaCaT, endogenous BAG-1 is primarily nuclear and increases with confluence. Both transient and stable p36-Bag-1 overexpression resulted in increased cellular cohesion. Stable transfection of either of the three human BAG-1 isoforms p36-Bag-1 (BAG-1S), p46-Bag-1 (BAG-1M) and p50-Bag-1 (BAG-1L) inhibited growth and wound closure in serum-containing medium. However, in response to hepatocyte growth factor (HGF) in serum-free medium, BAG-1S/M reduced communal motility and colony scattering, but BAG-1L did not. In the presence of HGF, p36-Bag-1 transfectants retained proliferative response to HGF with no change in ERK1/2 activation. However, the cells retained E-cadherin localisation at cell-cell junctions and exhibited pronounced cortical actin. Point mutations in the BAG domain showed that BAG-1 inhibition of motility is independent of its function as a chaperone regulator. These findings are the first to suggest that BAG-1 plays a role in regulating cell-cell adhesion and suggest an important function in epidermal cohesion.

Hsp40 molecules that target to the ubiquitin-proteasome system decrease inclusion formation in models of polyglutamine disease.

We studied the ability of heat shock, DnaJ-like-1 (HSJ1) proteins (which contain DnaJ and ubiquitin-interacting motifs) to reduce polyglutamine-mediated inclusion formation. The experiments demonstrated that expression of heat shock protein 70 (hsp70), hsp40, HSJ1a, and HSJ1b significantly reduced protein inclusion formation in a model of spinal and bulbar muscular atrophy (SBMA). HSJ1a also mediated a significant decrease in the number of inclusions formed in a primary neuronal model of protein aggregation. Studies to elucidate the mechanisms underlying these reductions showed that hsp70 and hsp40 increased chaperone-mediated refolding. In contrast, expression of HSJ1 proteins did not promote chaperone activity but caused an increase in ubiquitylation. Furthermore, HSJ1a was associated with a ubiquitylated luciferase complex, and in the presence of HSJ1a but not an HSJ1a UIM mutant (HSJ1a-deltaUIM) there was a reduction in luciferase protein levels. Together these results show that HSJ1 proteins mediated an increase in target protein degradation via the ubiquitin-proteasome system (UPS). We also found that the expression of HSJ1a significantly decreased the number of neurons containing inclusions in an in vivo model of polyglutamine disease. These findings indicate that targeted modification of the UPS to facilitate degradation of misfolded proteins may represent a highly effective therapeutic avenue for the treatment of polyglutamine disease.

Hsp40 Molecules That Target to the Ubiquitin-proteasome System Decrease Inclusion Formation in Models of Polyglutamine Disease.

We studied the ability of heat shock, DnaJ-like-1 (HSJ1) proteins (which contain DnaJ and ubiquitin-interacting motifs) to reduce polyglutamine-mediated inclusion formation. The experiments demonstrated that expression of heat shock protein 70 (hsp70), hsp40, HSJ1a, and HSJ1b significantly reduced protein inclusion formation in a model of spinal and bulbar muscular atrophy (SBMA). HSJ1a also mediated a significant decrease in the number of inclusions formed in a primary neuronal model of protein aggregation. Studies to elucidate the mechanisms underlying these reductions showed that hsp70 and hsp40 increased chaperone-mediated refolding. In contrast, expression of HSJ1 proteins did not promote chaperone activity but caused an increase in ubiquitylation. Furthermore, HSJ1a was associated with a ubiquitylated luciferase complex, and in the presence of HSJ1a but not an HSJ1a UIM mutant (HSJ1a-ΔUIM) there was a reduction in luciferase protein levels. Together these results show that HSJ1 proteins mediated an increase in target protein degradation via the ubiquitin-proteasome system (UPS). We also found that the expression of HSJ1a significantly decreased the number of neurons containing inclusions in an in vivo model of polyglutamine disease. These findings indicate that targeted modification of the UPS to facilitate degradation of misfolded proteins may represent a highly effective therapeutic avenue for the treatment of polyglutamine disease.

Radiation-induced life shortening in neonatally thymectomized germ-free mice.

Radiation in sufficient amounts is carcinogenic, immunosuppressive, and results in a reduced life span. Similar consequences follow neonatal thymectomy (nTx) in some strains of rodents. The tumorigenic effects of irradiation appear to be partly mediated via suppression of the thumus-dependent portion of the immune response. Our purpose was to determine whether a similar relationship exists for radiation-induced accelerated aging. Female germ-free Charles River mice had neonatal or sham thymectomies within 24 hours of birth. Half of each group was exposed to 700 rads at 6 weeks of age. When mice with histologically malignant tumors were excluded, the combined life-shortening effects of nTx and irradiation were less pronounced than the sum of the individual effects. This suggests that some of the decreased longevity associated with irradiation may be mediated by T-cell injury.

Effects of whole-body irradiation on neonatally thymectomized mice. Incidence of benign and malignant tumors.

The individual and combined effects of neonatal thymectomy and whole-body irradiation on the prevalence of benign and malignant tumors in germ-free female mice of the Charles Rivers line were studied to determine if a portion of the tumorigenic effects of irradiation can be attributed to injury of the thymic-dependent component of the immune response. Neonatal thymectomy increased a) the incidence of benign and malignant tumors and b) the prevalence of multiple primary neoplasms in an individual mouse. Whole-body exposure to 700 rad at 6 weeks of age further increased th incidence of tumors, but the relative magnitude of this increase was less pronounced than in sham-operated controls. Thus, the cumulative effects of thymectomy plus irradiation are less pronounced than the sum of the individual effects. One of several possible explanations for this observation is that a portion of the carcinogenic effects of whole-body irradiation is mediated by suppression of the thymic-dependent component of the immune response.

Radiosensitivity of T and B lymphocytes. IV. Effect of whole body irradiation upon various lymphoid tissues and numbers of recirculating lymphocytes.

Groups of 10-week-old female CBA/J mice were exposed in whole body fashion to 0,5,50, and 500 rads and sacrificed in serial fashion 1,3,5,7,9,15, and 30 days after irradiation for morphologic evaluation of thymus, spleen, lymph node, and Peyer's patch, and assessment of the relative numbers of thymus-derived (T) and bone marrow-derived (B) cells in these tissues. The absolute and relative numbers of recirculating T and B cells mobilizable by thoracic duct cannulation were also determined and compared with similar determinations with respect to peripheral blood lymphocytes. B cell depletion occurred more quickly and was more pronounced in spleen and lymph node than T cell depletion at all three exposure doses. Depletion of T and B cells was roughly equal in peripheral blood and thoracic duct lymph. When present, regeneration of the T cell component occurred more rapidly than did B cell restoration. The latter often was incomplete at the time of the final sacrifice (day 30). PHA-responsive and Con A-responsive cells also appeared to differ with respect to the kinetics of cell death after whole body irradiation.

Computer analysis of defined populations of lymphocytes irradiated in vitro. II. Analysis of thymus-dependent versus bone marrow-dependent cells.

Three uniform populations of T and B cells exposed to varying amounts of x-irradiation are examined utilizing computer-assisted morphometric analysis. These populations are: thoracic duct lymphocytes (TDL) from congenitally athymic (nude) mice (B cells); TDL from CBA mice treated with anti-Ig plus complement (T cells); and computer-selected untreated T cells from CBA TDL. Irradiated B cells show a more even dispersion of the nuclear chromatin and a dose-dependent increase in relative nuclear area beginning with the lowest dose evaluated (50 rads); no significan change in total optical density (OD) is demonstrable over the dose range evaluated (0 to 2000 rads). Anti-Ig-treated irradiated T cells demonstrate an initial shift toward lower OD values as a function of dose followed by a marked rise of OD values at 2000 rads, where numerous densely staining Feulgen-positive aggregates are identified. The relative nuclear area of this cell population also shows a biphasic response to radiation injury with an initial increase at the lower dose levels followed by a progressive decline to approximate control levels at 2000 rads. This effect is mirrored by the alteration in total OD which, after a decrease at low dose levels, approximates control values at 2000 rads. The computer-selected T cells show little change in OD values at the low-dose levels but show a marked increase in the more densely staining Feulgen-positive material following 2000 rads. This population reveals no apparent change in either relative nuclear area or total OD as a function of dose. Thus, untreated computer-selected T cells exhibit remarkably little evidence, morphologically, of radiation injury of doses associated with pronounced alterations on the part of B cells. In addition, treatment of a mixed cell population (CBA TDL) with anti-Ig plus complement to remove the B cells appears to alter the response of the residual T cells to radiation injury. These results, in conjunction with recent evidence to support the concept that T cells possess surface Ig, suggest that an Ig-anti-Ig interaction may alter the radiosensitivity of T cells.

Computer analysis of defined populations of lymphocytes irradiated in vitro: I. Evaluation of murine thoracic duct lymphocytes.

Computer-assisted morphometric analysis of murine lymphocytes obtained by thoracic duct cannulation demonstrates two populations of cells; the larger population (73 percent) appears to be thymus-derived and the remaining 27 percent is of bone marrow origin. Following exposure to varying amounts of x-radiation, morphologic alterations in both populations are evident. The smaller cell populations are evident. The smaller cell population exhibits some of these changes at lower dose levels than does the larger population. In addition, the character of the radiation-induced changes appears to be different for the two populations of lymphocytes. After 500 rad, the nuclei of the larger population appears unchanged; the nuclei of the population representing 27 percent of the cells have become enlarged and vacuolated and are thought to be edematous. After 2000 rad, the nuclei of the larger population appear pyknotic with coarsely clumped chromatin. In the examined set of cells, the smaller population could no longer be detected after 2000 rad. Such disparate responses to radiation-induced injury may correlate with known differences in immunologic function which serve to distinguish thymic-dependent and bone marrow-derived small lymphocytes.