The Journal of biological chemistry
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Vascular endothelial growth factor (VEGF), a potent angiogenic mitogen, plays a crucial role in angiogenesis under various pathophysiological conditions. We have recently demonstrated that VEGF(165), one of the VEGF isoforms, binds connective tissue growth factor (CTGF) and that its angiogenic activity is inhibited in the VEGF(165). CTGF complex form (Inoki, I., Shiomi, T., Hashimoto, G., Enomoto, H., Nakamura, H., Makino, K., Ikeda, E., Takata, S., Kobayashi, K. and Okada, Y. (2002) FASEB J. 16, 219-221). ⋯ Recovery of angiogenic activity was further confirmed by in vivo angiogenesis assay using a Matrigel injection model in mice. These results demonstrate for the first time that CTGF is a substrate of MMPs and that the angiogenic activity of VEGF(165) suppressed by the complex formation with CTGF is recovered through the selective degradation of CTGF by MMPs. MMPs may play a novel role through CTGF degradation in VEGF-induced angiogenesis during embryonic development, tissue maintenance, and/or pathological processes of various diseases.
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While classically viewed as a prototypic G(s) and adenylyl cyclase-coupled G protein-coupled receptor, recent studies have indicated that some aspects of beta(2)-adrenergic receptor (beta(2)-AR) signaling are inhibited by pertussis toxin, indicating that they are mediated by G(i)/G(o) proteins. These signals include activation of ERK MAPKs and Akt activation, as well as hypertrophic and anti-apoptotic pathways in cardiac myocytes. Studies in cultured cells have suggested the hypothesis that protein kinase A (PKA)-mediated phosphorylation of the beta(2)-AR regulates its coupling specificity with respect to G(s) and G(i). ⋯ PKA-mediated phosphorylation of the beta(2)-AR significantly decreased its ability to couple to G(s), while simultaneously dramatically increasing its ability to couple to G(i). These results are reproduced when a purified recombinant Ser --> Asp mutant beta(2)-AR is tested, whereas the Ser --> Ala receptor resembles the unphosphorylated wild type. These results provide strong experimental support for the idea that PKA-mediated phosphorylation of the beta(2)-adrenergic receptor switches its predominant coupling from G(s) to G(i).
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We have reported previously (Michikawa, M., Fan, Q.-W., Isobe, I., and Yanagisawa, K. (2000) J. Neurochem. 74, 1008-1016) that exogenously added recombinant human apolipoprotein E (apoE) promotes cholesterol release in an isoform-dependent manner. However, the molecular mechanism underlying this isoform-dependent promotion of cholesterol release remains undetermined. ⋯ The molar ratios of cholesterol to apoE in the HDL fraction of the culture media of apoE3- and apoE4-expressing astrocytes were 250 +/- 6.0 and 119 +/- 5.1, respectively. These data indicate that apoE3 has an ability to generate similarly sized lipid particles with less number of apoE molecules than apoE4, suggesting that apoE3-expressing astrocytes can supply more cholesterol to neurons than apoE4-expressing astrocytes. These findings provide a new insight into the issue concerning the putative alteration of apoE-related cholesterol metabolism in Alzheimer's disease.
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The N-terminal domains of the lung collectins, surfactant proteins A (SP-A) and D (SP-D), are critical for surfactant phospholipid interactions and surfactant homeostasis, respectively. To further assess the importance of lung collectin N-terminal domains in surfactant structure and function, a chimeric SP-D/SP-A (D/A) gene was constructed by substituting nucleotides encoding amino acids Asn(1)-Ala(7) of rat SP-A with the corresponding N-terminal sequences from rat SP-D, Ala(1)-Asn(25). Recombinant D/A migrated as a 35-kDa band on reducing SDS-PAGE and as a ladder of disulfide-linked multimers under nonreducing conditions. ⋯ Surfactant isolated from SP-A(-/-,D/A) mice exhibited elevated surface tension both in the presence and absence of plasma inhibitors, but whole lung compliance of the SP-A(-/-,D/A) animals was not different from the SP-A(-/-) littermates. Lung-specific overexpression of D/A in the SPD(-/-) mouse resulted in hetero-oligomer formation with mouse SP-A and did not correct the air space dilation or phospholipidosis that occurs in the absence of SP-D. These studies indicate that the N terminus of SP-D 1) can functionally replace the N terminus of SP-A for lipid aggregation and tubular myelin formation, but not for surface tension lowering properties of SP-A, and 2) is not sufficient to reverse the structural and metabolic pulmonary defects in the SP-D(-/-) mouse.
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Exercise increases glucose transport in muscle by activating 5'-AMP-activated protein kinase (AMPK), but subsequent events are unclear. Presently, we examined the possibility that AMPK increases glucose transport through atypical protein kinase Cs (aPKCs) by activating proline-rich tyrosine kinase-2 (PYK2), ERK pathway components, and phospholipase D (PLD). In mice, treadmill exercise rapidly activated ERK and aPKCs in mouse vastus lateralis muscles. ⋯ Similarly, in L6 myotubes, (a) AICAR activated PYK2, ERK, PLD, and aPKCs; (b) effects of AICAR on ERK were inhibited by genistein, PD98059, and expression of dominant-negative PYK2; (c) effects of AICAR on PLD were inhibited by MEK1 inhibitor UO126; (d) effects of AICAR on aPKCs were inhibited by genistein, PD98059, 1-butanol, and expression of dominant-negative forms of PYK2, GRB2, SOS, RAS, RAF, and ERK; and (e) effects of AICAR on 2DOG uptake/GLUT4 translocation were inhibited by genistein, PD98059, UO126, 1-butanol, cell-permeable myristoylated PKC-zeta pseudosubstrate, and expression of kinase-inactive RAF, ERK, and PKC-zeta. AMPK activator dinitrophenol had effects on ERK, aPKCs, and 2-DOG uptake similar to those of AICAR. Our findings suggest that effects of exercise on glucose transport that are dependent on AMPK are mediated via PYK2, the ERK pathway, PLD, and aPKCs.