Severe muscular dystrophy in mice that lack dystrophin and alpha7 integrin.

The dystrophin glycoprotein complex links laminin in the extracellular matrix to the cell cytoskeleton. Loss of dystrophin causes Duchenne muscular dystrophy, the most common human X-chromosome-linked genetic disease. The alpha7beta1 integrin is a second transmembrane laminin receptor expressed in skeletal muscle. Mutations in the alpha7 integrin gene cause congenital myopathy in humans and mice. The alpha7beta1 integrin is increased in the skeletal muscle of Duchenne muscular dystrophy patients and mdx mice. This observation has led to the suggestion that dystrophin and alpha7beta1 integrin have complementary functional and structural roles. To test this hypothesis, we generated mice lacking both dystrophin and alpha7 integrin (mdx/alpha7(-/-)). The mdx/alpha7(-/-) mice developed early-onset muscular dystrophy and died at 2-4 weeks of age. Muscle fibers from mdx/alpha7(-/-) mice exhibited extensive loss of membrane integrity, increased centrally located nuclei and inflammatory cell infiltrate, greater necrosis and increased muscle degeneration compared to mdx or alpha7-integrin null animals. In addition, loss of dystrophin and/or alpha7 integrin resulted in altered expression of laminin-alpha2 chain. These results point to complementary roles for dystrophin and alpha7beta1 integrin in maintaining the functional integrity of skeletal muscle.

Phosphoinositide-dependent kinase 1 and p21-activated protein kinase mediate reactive oxygen species-dependent regulation of platelet-derived growth factor-induced smooth muscle cell migration.

Smooth muscle cell migration in response to platelet-derived growth factor (PDGF) is a key event in several vascular pathologies, including atherosclerosis and restenosis. PDGF increases intracellular levels of reactive oxygen species (ROS) in vascular smooth muscle cells (VSMCs), but the ROS sensitivity of migration and of the signaling pathways leading to migration are largely unknown. In VSMCs, PDGF dose-dependently increased migration compared with nonstimulated cells, with a maximum increase at 10 ng/mL. Pretreatment with the antioxidant N-acetyl-cysteine, the flavin-containing enzyme inhibitor diphenylene iodonium, or the glutathione peroxidase mimetic ebselen significantly attenuated migration (PDGF alone, 5.0+/-1.1-fold; NAC, 1.8+/-0.2-fold; diphenylene iodonium, 1.4+/-0.3-fold migration; and ebselen, 2.0+/-0.5-fold migration), as did overexpression of catalase. Pretreatment of VSMCs with the Src inhibitor PP1 or dominant-negative Rac adenovirus significantly inhibited migration, but only Src activation was attenuated by ROS inhibitors. Phosphorylation of the Src- and Rac-effector p21-activated protein kinase (PAK) 1 on Thr423 (the phosphoinositide-dependent kinase-1 [PDK1] site) was attenuated by ROS inhibition, and infection of VSMCs with dominant-negative PAK1 adenovirus attenuated migration. Moreover, kinase-inactive K111N-PDK1 inhibited PAK1 phosphorylation on Thr423, and both K111N-PDK1 and Y9F-PDK1 significantly inhibited VSMC migration. PDK1 tyrosine phosphorylation was also ROS dependent. These data indicate that PDGF-induced VSMC migration is ROS dependent and identify the Src/PDK1/PAK1 signaling pathway as an important ROS-sensitive mediator of migration. Such information is critical to understanding the role of ROS in vascular diseases in which migration of VSMCs is an important component.

p38 MAPK and NF-kappaB mediate COX-2 expression in human airway myocytes.

We have previously demonstrated that p38 and extracellular signal-regulated protein kinase (ERK) mitogen-activated protein kinases (MAPK) are components of proinflammatory induced cytokine expression in human airway myocytes. The experiments described here further these studies by examining p38 MAPK and NF-kappaB regulation of cyclooxygenase-2 (COX-2) expression in response to a complex inflammatory stimulus consisting of 10 ng/ml interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha), and interferon (IFN)-gamma. COX-2 expression was induced with this stimulus in a time-dependent manner, with maximal expression seen 12-20 h after treatment. Semiquantitative RT-PCR and immunoblotting experiments demonstrate decreased COX-2 expression following treatment with the p38 MAPK inhibitor SB-203580 (25 microM) or the proteosome inhibitor MG-132 (1 microM). SB-203580 did not affect cytokine-stimulated IkappaBalpha degradation, NF-kappaB nuclear binding activity, or NF-kappaB-dependent signaling from the COX-2 promoter, indicating that p38 MAPK and NF-kappaB may affect COX-2 expression via separate signaling pathways. SB-203580, but not MG-132, also increased the initial rate of COX-2 mRNA decay, indicating p38 MAPK, but not NF-kappaB, participates in the regulation of COX-2 mRNA stability. These findings suggest that although p38 MAPK and NF-kappaB signaling regulate steady-state levels of COX-2 expression, p38 MAPK additionally affects stability of COX-2 mRNA in cytokine-stimulated human airway myocytes.