Bcl-2 rescues motoneurons from early cell death in the cervical spinal cord of the chicken embryo

Motoneurons (MNs) in the cervical spinal cord of the chicken embryo undergo programmed cell death (PCD) between embryonic day (E) 4 and E5. The intracellular molecules regulating this early phase of PCD remain unknown. Here we show that introduction of Bcl-2 by a replication-competent avian retroviral vector prevented MN degeneration at E4.5, whereas the expression of the green fluorescent protein (GFP) was ineffective. Bcl-2 expression did not affect the number of Islet-1/2-positive MNs at the onset of cell death (E4). However, when examined at the end of the cell death period (E5.5), the number of Islet-1/2-positive MNs was clearly increased in Bcl-2-transfected embryos compared with control and GFP-transfected embryos. Activation of caspase-3, which is normally observed in this early MN death, was also prevented by Bcl-2. Thus, MNs in the cervical spinal cord appear to use intracellular pathway(s) for early PCD that is responsive to Bcl-2.     

Gads/Grb2-mediated association with LAT is critical for the inhibitory function of Gab2 in T cells

A docking protein, Gab2, is recruited to the vicinity of the TCR complex and inhibits downstream signaling by interaction with negative regulators. However, the molecular mechanisms of this recruitment remain unclear. We have found that Gab2 associates with LAT upon TCR stimulation and that LAT is essential for Gab2 phosphorylation. By analysis of several Gab2 mutants, the c-Met binding domain (MBD) of Gab2 was found to be both necessary and sufficient for stimulation-induced LAT binding. Within the MBD domain, a novel Grb2 SH3 binding motif, PXXXR, is critical for constitutive association with Gads/Grb2. Through this association, Gab2 is recruited to the lipid raft after TCR ligation and exerts inhibitory function. The in vivo significance of this association is illustrated by the fact that T-cell responses are impaired in transgenic mice expressing wild-type Gab2 but not in mice expressing mutant Gab2 lacking the motif. Furthermore, T cells from Gab2-deficient mice showed enhanced proliferative responses upon TCR stimulation. These results indicate that Gads/Grb2-mediated LAT association is critical for the inhibitory function of Gab2, implying that Gab2 induced in stimulated T cells may exert an efficient negative feedback loop by recruiting inhibitory molecules to the lipid raft and competing with SLP-76 through Gads binding.     

A novel interaction between thyroid hormones and 1,25(OH)(2)D(3) in osteoclast formation

Thyroid hormones enhance osteoclast formation and their excess is an important cause of secondary osteoporosis. 3,5,3' -Triiodo-L-thyronine (T3) induced the mRNA expression of receptor activator of nuclear factor-kappa B ligand (RANKL), which is a key molecule in osteoclast formation, in primary osteoblastic cells (POB). This effect was amplified in the copresence of 1 alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). Although T3 alone did not induce octeoclasts in coculture of bone marrow cells with POB, T3 enhanced 1,25(OH)(2)D(3)-induced osteoclast formation. Thyroxine (T4) also enhanced 1,25(OH)(2)D(3)-induced osteoclast formation. These data suggested that T4 was locally metabolized to T3 for its action, since T4 is a prohormone with little hormonal activity. The mRNA expression of type-2 iodothyronine deiodinase (D2), which is responsible for maintaining local T3 concentration, was induced by 1,25(OH)(2)D(3) dose- and time-dependently. Our data would facilitate our understanding of the mechanism of osteoclast formation by thyroid hormones and suggest a novel interaction between thyroid hormones and 1,25(OH)(2)D(3).     

Differential regulation of diacylglycerol kinase isozymes in cardiac hypertrophy

To examine the involvement of diacylglycerol kinase (DGK) and phosphatidic acid phosphatase (PAP) in pressure overloaded cardiac hypertrophy, rats were subjected to either ascending aortic banding for 3, 7, and 28 days or sham operation. In comparison with sham-operated rats, the left ventricular (LV) weight of the aortic-banded rats increased progressively. At 28 days after surgery, the expression of DGKepsilon mRNA but not DGKzeta or PAP2b mRNA in the LV myocardium significantly decreased in the aortic-banded rats compared with the sham-operated rats. DGKzeta protein in the LV myocardium translocated from the particulate to the cytosolic compartment in the aortic-banded rats. Furthermore, the myocardial content of 1,2-diacylglycerol and PKCdelta protein expression in the particulate fraction of the LV myocardium significantly increased in aortic-banded rats compared with sham-operated rats. These results suggest that DGKepsilon and DGKzeta play distinct roles in the development of pressure overloaded cardiac hypertrophy and that the two isozymes are differentially regulated.     

Regulation of Src kinase activity during Xenopus oocyte maturation

Expression of constitutively active Src protein tyrosine kinase in Xenopus oocytes has been shown to accelerate oocyte maturation suggesting that Src may be involved in meiotic progression. However, meiotic regulation of endogenous Src kinase in oocytes has not been investigated in detail. To address this problem, we measured the activity, expression level, and phosphorylation state of the endogenous Xenopus Src (xSrc) and overexpressed xSrc mutants in the process of progesterone-induced oocyte maturation. We found that the enzyme is first transiently activated in the plasma membrane-containing fraction of oocytes within 3 min of progesterone administration. This event represents one of the earliest responses of oocytes to the hormone and should be related to triggering some early signaling pathways of maturation. Thereafter, xSrc activity increases again at the time of germinal vesicle breakdown (GVBD) and remains elevated till the completion of maturation. This elevation of xSrc activity is associated with a 2-fold increase of xSrc protein content in the absence of change in its specific activity and xSrc mRNA content. No significant changes in the phosphorylation state of C-terminal regulatory phosphotyrosine can be registered either in endogenous xSrc or in overexpressed kinase-negative and wild-type xSrc proteins during maturation. Altogether, these results indicate that upregulation of xSrc in the meiotic metaphase occurs at the translation level. We also demonstrate here that the expression of constitutively active xSrc in Xenopus oocytes is accompanied by the activation of mitogen-activated protein kinase (MAPK). Our data suggest that the Src kinase acts through the MAPK pathway to accelerate oocyte maturation.     

Wnt signaling controls the timing of oligodendrocyte development in the spinal cord

During spinal cord development, oligodendrocytes are generated from a restricted region of the ventral ventricular zone and then spread out into the entire spinal cord. These events are controlled by graded inductive and repressive signals derived from a local organizing center. Sonic hedgehog was identified as an essential ventral factor for oligodendrocyte lineage specification, whereas the dorsal cue was less clear. In this study, Wnt proteins were identified as the dorsal factors that directly inhibit oligodendrocyte development. Wnt signaling through a canonical beta-catenin pathway prevents its differentiation from progenitor to an immature state. Addition of rmFz-8/Fc, a Wnt antagonist, increased the number of immature oligodendrocytes in the spinal cord explant culture, demonstrating that endogenous Wnt signaling controls oligodendrocyte development.     

Collaborated regulation of female-specific murine Cyp3a41 gene expression by growth and glucocorticoid hormones

CYP3A41 is a female-specific major CYP3A in mouse livers. Adrenalectomy decreased expression of CYP3A41 as well as CYP3A11, another major CYP3A, and dexamethasone (DEX) restored the decreased expression. Hypophysectomy completely abolished CYP3A41 expression and growth hormone (GH) replacement only slightly restored the expression. Treatment with DEX alone did not induce expression of either CYP3A41 or CYP3A11 in hypophysectomized mice. However, combined treatment with GH and DEX strongly induced expression of CYP3A41 but not CYP3A11. In primary cultured mouse hepatocytes, DEX induced expression of both CYP3A41 and CYP3A11, and DEX-inducible expression of CYP3A41 was suppressed by RU486, a potent antiglucocorticoid. In contrast, RU486 by itself enhanced basal expression of CYP3A11 mRNA, while it showed no inhibitory effect on DEX-inducible expression. These observations indicate that glucocorticoids may participate in the GH-dependent control of the Cyp3a41 gene expression, probably mediated via the glucocorticoid receptor, which may be different from that of the Cyp3a11 gene expression.     

Molecular cloning and characterization of Bifidobacterium bifidum 1,2-alpha-L-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95)

A genomic library of Bifidobacterium bifidum constructed in Escherichia coli was screened for the ability to hydrolyze the alpha-(1-->2) linkage of 2'-fucosyllactose, and a gene encoding 1,2-alpha-l-fucosidase (AfcA) was isolated. The afcA gene was found to comprise 1,959 amino acid residues with a predicted molecular mass of 205 kDa and containing a signal peptide and a membrane anchor at the N and C termini, respectively. A domain responsible for fucosidase activity (the Fuc domain; amino acid residues 577 to 1474) was localized by deletion analysis and then purified as a hexahistidine-tagged protein. The recombinant Fuc domain specifically hydrolyzed the terminal alpha-(1-->2)-fucosidic linkages of various oligosaccharides and a sugar chain of a glycoprotein. The stereochemical course of the hydrolysis of 2'-fucosyllactose was determined to be inversion by using (1)H nuclear magnetic resonance. The primary structure of the Fuc domain exhibited no similarity to those of any glycoside hydrolases (GHs) but showed high similarity to those of several hypothetical proteins in a database. Thus, it was revealed that the AfcA protein constitutes a novel inverting GH family (GH family 95).     

An autocrine/paracrine loop linking keratin 14 aggregates to tumor necrosis factor alpha-mediated cytotoxicity in a keratinocyte model of epidermolysis bullosa simplex

Epidermolysis bullosa simplex (EBS) is a blistering cutaneous disease featuring protein aggregates. Here we investigate the molecular mechanisms linking protein aggregates to cell death in a cellular model of EBS in which HaCaT keratinocytes are transfected with plasmids expressing various mutant forms of keratin 14 (K14). In HaCaT cells, mutant K14 was found to form ubiquitinated protein aggregates that suppressed 20 S proteasome function instead of being degraded by 20 S proteasome. Keratinocytes with mutant K14-induced phosphorylation of the stress-activated kinase c-Jun, as well as up-regulation of unfolding protein Bip, indicates induction of endoplasmic reticulum stress. HaCaT cells were susceptible to apoptosis by activation of caspases-3, and -8, but not caspase-9 or -12. Tumor necrosis factor-alpha (TNFalpha) in the culture medium was increased in keratinocytes with mutant K14 compared with wild K14, and the addition of neutralizing anti-TNFalpha antibody to the culture medium rescued keratinocytes from cell death. Thus, TNFalpha release and the subsequent activation of the TNFalpha receptor by an autocrine/paracrine pathway links protein aggregates to cell death in this keratinocyte EBS cellular model. Furthermore, mutation in K14 reduced its affinity to TNFalpha receptor-associated death domain (TRADD), suggesting that the susceptibility of keratinocytes to caspase-8-mediated apoptosis is increased in mutated K14 because of impairment of the cytoprotective mechanism mediated by K14-TRADD interaction.