Reduction of IKKα Expression Promotes Chronic Ultraviolet B Exposure-Induced Skin Inflammation and Carcinogenesis

Ultraviolet B light (UVB) is a common cause of human skin cancer. UVB irradiation induces mutations in the tumor suppressor p53 gene as well as chronic inflammation, which are both essential for UVB carcinogenesis. Inhibitor of nuclear factor κB kinase-α (IKKα) plays an important role in maintaining skin homeostasis, and expression of IKKα was found to be down-regulated in human and murine skin squamous cell carcinomas. However, the role of IKKα in UVB skin carcinogenesis has not been investigated. Thus, here we performed UVB carcinogenesis experiments on Ikkα^+/+ and Ikkα^+/− mice. Ikkα^+/− mice were found to develop a twofold greater number of skin tumors than Ikkα^+/+ mice after chronic UVB irradiation. In addition, tumor latency was significantly shorter and tumors were bigger in Ikkα^+/− than in Ikkα^+/+ mice. At an early stage of carcinogenesis, an increase in UVB-induced p53 mutations as well as macrophage recruitment and mitogenic activity, and a decrease in UVB-induced apoptosis, were detected in Ikkα^+/− compared with those in Ikkα^+/+ skin. Also, reduction of IKKα levels in keratinocytes up-regulated the expression of monocyte chemoattractant protein-1 (MCP-1/CCL2), TNFα, IL-1, and IL-6, and elevated macrophage migration, which might promote macrophage recruitment and inflammation. Therefore, these findings suggest that reduction of IKKα expression orchestrates UVB carcinogen, accelerating tumorigenesis.Ultraviolet B (UVB) irradiation induces DNA damage. The tumor suppressor gene p53 is an UVB target, and human cutaneous squamous cell carcinoma (SCC) cells contain p53 mutations.¹ Skin cells can harbor UVB-induced p53 mutations for decades before the onset of human SCC, however,^2,3 underscoring the importance of coactivators in skin tumorigenesis. In mice, p53 mutations are an early genetic event in UVB skin carcinogenesis, which recapitulates the process of human SCC development.^4,5 The p53 mutations have been proposed to be important for UVB carcinogenesis because prevention of these mutations prevents skin tumor development, and the number of p53 mutation-positive keratinocytes correlates with the number of skin tumors in mice.^6,7,8,9 UVB exposure also induces chronic inflammation, cell proliferation, oxidant stress, and immunosuppression, which essentially facilitate UVB carcinogenesis.^10,11 Particularly, chronic inflammation can create a microenvironment that is prone to cell proliferation and DNA damage, thereby promoting tumor development.¹²On the other hand, UVB exposure also elicits protective responses, such as cell cycle arrest, DNA repair, and apoptosis, which reduce UVB-induced damage.^7,10,11 Previously, it was reported that mice lacking p53 were defective in inducing apoptosis after UVB irradiation and thus had more skin tumors than wild-type mice did.^13,14 Mice lacking Fas ligand had defects in apoptosis, which increased numbers of cells containing UVB-induced p53 mutations, and the mutant mice were more susceptible to UVB-induced skin tumors than wild-type mice.^5,8 Therefore, the different defects in inducing protective responses against UVB-induced damage can amplify the severity of the cancer cause, thereby accelerating carcinogenesis.Inhibitor of nuclear factor κB kinase-α (IKKα) is required for embryonic skin development in mice.^15,16 Several laboratories have reported the down-regulated expression and altered localization of IKKα protein as well as deletions and mutations in the Ikkα gene in human SCCs of the skin, lung, esophagus, and head and neck.^{17,18,19,20,21} In particular, IKKα deletion in keratinocytes was found to elevate an autocrine loop activity of epidermal growth factor receptor (EGFR), Ras, extracellular signal-related kinase (ERK), EGFR ligands, and the ligands’ activators and induced spontaneous skin SCCs in mice.²² Reduction of IKKα expression was found to promote chemical carcinogen-induced skin tumorigenesis.²³ Also, IKKα is an UVB-induced gene, and the defect in IKKα function was linked to inflammation.^24,25 UVB is a very common cause of human skin cancer; however, the role of IKKα in skin UVB carcinogenesis is largely unknown. Thus, here we examined the effect of reduced IKKα expression on UVB skin carcinogenesis in Ikkα^+/− and Ikkα^+/+ mice. Ikkα^+/− mice were significantly susceptible to UVB skin carcinogenesis than were Ikkα^+/+ mice. Because the tumor latency was significantly shorter and many more tumors were found in Ikkα^+/− mice than in Ikkα^+/+ mice, we analyzed the early events during UVB skin carcinogenesis. This study provided the first evidence showing the importance of IKKα in UVB skin carcinogenesis.     

Phenolic Compounds Prevent Alzheimer’s Pathology through Different Effects on the Amyloid-β Aggregation Pathway

Inhibition of amyloid-β (Aβ) aggregation is an attractive therapeutic strategy for Alzheimer’s disease (AD). Certain phenolic compounds have been reported to have anti-Aβ aggregation effects in vitro. This study systematically investigated the effects of phenolic compounds on AD model transgenic mice (Tg2576). Mice were fed five phenolic compounds (curcumin, ferulic acid, myricetin, nordihydroguaiaretic acid (NDGA), and rosmarinic acid (RA)) for 10 months from the age of 5 months. Immunohistochemically, in both the NDGA- and RA-treated groups, Aβ deposition was significantly decreased in the brain (P < 0.05). In the RA-treated group, the level of Tris-buffered saline (TBS)-soluble Aβ monomers was increased (P < 0.01), whereas that of oligomers, as probed with the A11 antibody (A11-positive oligomers), was decreased (P < 0.001). However, in the NDGA-treated group, the abundance of A11-positive oligomers was increased (P < 0.05) without any change in the levels of TBS-soluble or TBS-insoluble Aβ. In the curcumin- and myricetin-treated groups, changes in the Aβ profile were similar to those in the RA-treated group, but Aβ plaque deposition was not significantly decreased. In the ferulic acid-treated group, there was no significant difference in the Aβ profile. These results showed that oral administration of phenolic compounds prevented the development of AD pathology by affecting different Aβ aggregation pathways in vivo. Clinical trials with these compounds are necessary to confirm the anti-AD effects and safety in humans.Alzheimer’s disease (AD) is the most common form of dementia, resulting in deterioration of cognitive function and behavioral changes.¹ One of the pathological hallmarks of AD is extracellular deposits of aggregated amyloid-β protein (Aβ) in the brain parenchyma (senile plaques) and cerebral blood vessels (cerebral amyloid angiopathy (CAA)).¹ Deposition of high levels of fibrillar Aβ in the AD brain is associated with loss of synapses, impairment of neuronal functions, and loss of neurons.^2,3,4,5 Aβ was sequenced from meningeal vessels and senile plaques of AD patients and individuals with Down’s syndrome.^6,7,8 The subsequent cloning of the gene encoding the β-amyloid precursor protein and its localization to chromosome 21,^9,10,11,12 coupled with the earlier recognition that trisomy 21 (Down’s syndrome) invariably leads to the neuropathology of AD,¹³ set the stage for the proposal that Aβ accumulation is the primary event in AD pathogenesis. In addition, certain mutations associated with familial AD have been identified within or near the Aβ region of the coding sequence of gene of the amyloid precursor proteins,^14,15 presenilin-1 and presenilin-2,¹⁶ which alter amyloid precursor protein metabolism through a direct effect on γ-secretase.^17,18 These findings set the stage for the proposal that Aβ aggregation is the primary event in AD pathogenesis and leading to the proposal that anti-Aβ aggregation is a strategy for AD therapy.^19,20 Furthermore, there have been recent reports^{21,22,23,24,25} that Aβ fibrils are not the only toxic form of Aβ for developing AD, and smaller species of aggregated Aβ, Aβ oligomers, may represent the primary toxic species in AD. Therefore, it is necessary to consider the inhibition of Aβ oligomer formation as well as Aβ fibrils for the treatment of AD.²⁶To date, it has been reported that various compounds inhibit the formation and extension of Aβ fibrils, as well as destabilizing Aβ fibrils in vitro.^{19,20,27,28,29,30,31,32,33,34,35,36} Among the reported compounds, several phenolic compounds, such as wine-related polyphenols (myricetin (Myr), morin, and tannic acid, and so on), curcumin (Cur), ferulic acid (FA), nordihydroguaiaretic acid (NDGA), and rosmarinic acid (RA) had especially strong anti-Aβ aggregation effects in vitro. Furthermore, it was shown recently that a commercially available grape seed polyphenolic extract, MegaNatural-Az, inhibited fibril formation, protofibril formation, and oligomerization of Aβ.³⁷ Moreover, MegaNatural-Az also reduced cerebral amyloid deposition as well as attenuating AD-type cognitive deterioration using transgenic mice.³⁸ In addition to these studies by the current authors, several other researchers have reported similar effects of phenolic compounds.^{26,39,40,41,42,43,44} First, Cur decreased cerebral Aβ plaque burden in vivo,^{39,40,41,42,44} and inhibited the formation of Aβ oligomers in vitro.^26,39 Second, epigallocatechin gallate efficiently inhibited fibril and oligomer formation of Aβ.⁴³ However, a very recent in vitro study²⁶ reported that Cur, Myr, and NDGA inhibited the formation of Aβ oligomers, but Cur and NDGA promoted the formation of Aβ fibrils. This indicated that the effects of these phenolic compounds on Aβ aggregation remain controversial. These different results may reflect different experimental conditions in these studies. To resolve this problem, a systematic in vivo study is required; however, few reports on the effects of phenolic compounds on Aβ aggregation in vivo have been published so far, except for reports about Cur.^{39,40,41,42,44}To elucidate the inhibitory effects of phenolic compounds on Aβ aggregation in vivo, several phenolic compounds, including Cur, FA, Myr, NDGA, and RA, were fed to AD model mice, and the cerebral plaque burden and formation of Aβ oligomers were compared systematically.     

Amyloid-Peptide Vaccinations Reduce β-Amyloid Plaques but Exacerbate Vascular Deposition and Inflammation in the Retina of Alzheimer’s Transgenic Mice

Alzheimer’s disease (AD) is pathologically characterized by accumulation of β-amyloid (Aβ) protein deposits and/or neurofibrillary tangles in association with progressive cognitive deficits. Although numerous studies have demonstrated a relationship between brain pathology and AD progression, the Alzheimer’s pathological hallmarks have not been found in the AD retina. A recent report showed Aβ plaques in the retinas of APPswe/PS1ΔE9 transgenic mice. We now report the detection of Aβ plaques with increased retinal microvascular deposition of Aβ and neuroinflammation in Tg2576 mouse retinas. The majority of Aβ-immunoreactive plaques were detected from the ganglion cell layer to the inner plexiform layer, and some plaques were observed in the outer nuclear layer, photoreceptor outer segment, and optic nerve. Hyperphosphorylated tau was labeled in the corresponding areas of the Aβ plaques in adjacent sections. Although Aβ vaccinations reduced retinal Aβ deposits, there was a marked increase in retinal microvascular Aβ deposition as well as local neuroinflammation manifested by microglial infiltration and astrogliosis linked with disruption of the retinal organization. These results provide evidence to support further investigation of the use of retinal imaging to diagnose AD and to monitor disease activity.Cerebral abnormalities including neuronal loss, neurofibrillary tangles, senile plaques with aggregated β-amyloid protein (Aβ) deposits, microvascular deposition of Aβ, and inflammation are well-known pathological hallmarks of Alzheimer’s disease (AD).^1,2,3 Despite the controversial evidence about the contribution of Aβ to the development of AD-related cognitive deficits, accumulation of toxic, aggregated forms of Aβ plays a crucial role in the pathogenesis of familial types of AD.^4,5 Overexpression of amyloid precursor protein (APP) in trisomy 21, altered APP processing resulting from mutations in APP, presenilin 1 (PS1), or 2 (PS2), and, as-of-yet unidentified other familial AD, related mutations, lead to Aβ deposition and Aβ plaques in the brain as well as cognitive abnormalities.^6,7 Therefore, to understand the molecular basis of amyloid protein deposition and to detect Aβ plaques in brain, parenchyma ante-mortem are currently among the most active areas of research in AD.Besides cognitive abnormalities, patients with AD commonly complain of visual anomalies, in particular, related to color vision,^8,9 spatial contrast sensitivity,¹⁰ backward masking,¹¹ visual fields,¹² and other visual performance tasks.^13,14,15,16 In addition to the damage and malfunction in the central visual pathways, retinal abnormalities such as ganglion cell degeneration,¹⁷ decreased thickness of the retinal nerve fiber layer,^18,19 and optic nerve degeneration^20,21 may, in part, account for AD-related visual dysfunction. Although intracellular Aβ deposition has been detected in both ganglion and lens fiber cells of patients with glaucoma, AD, or Down’s syndrome,^22,23,24,25 other typical hallmarks of AD have not yet been demonstrated. Interestingly, thioflavine-S-positive Aβ plaques were recently found in the retinal strata of APPswe/PS1ΔE9 transgenic mice²⁶ but not in the other animal models of AD. The current study used Tg2576 mice that constitutively overexpress APPswe and develop robust Aβ deposits in brain as well as cognitive abnormalities with aging.²⁷ We assessed the pathological changes in the retina of aged mice following different immunization schemes. We immunized Tg2576 with fibrillar Aβ42 and with a prefibrillar oligomer mimetic that gives rise to a prefibrillar oligomer-specific immune response. Both types of immunogens have been shown to be equally effective in reducing plaque deposition and inflammation in Tg2576 mouse brains.²⁸ In this study, we also used another prefibrillar oligomer mimetic antigen that uses the islet amyloid polypeptide (IAPP) instead of Aβ, but which gives rise to the same generic prefibrillar oligomer-specific immune response that also recognizes Aβ prefibrillar oligomers.²⁹ Aβ plaques and microvascular Aβ deposition were observed in the control Tg2576 mouse retinas. In contrast, Aβ and IAPP prefibrillar oligomer vaccinations differentially removed retinal Aβ deposits but exacerbated retinal amyloid angiopathy and inflammation as demonstrated by a significantly enhanced microglial infiltration and astrogliosis.     

Integrin-Mediated Transforming Growth Factor-β Activation,a Potential Therapeutic Target in Fibrogenic Disorders

A subset of integrins function as cell surface receptors for the profibrotic cytokine transforming growth factor-β (TGF-β). TGF-β is expressed in an inactive or latent form, and activation of TGF-β is a major mechanism that regulates TGF-β function. Indeed, important TGF-β activation mechanisms involve several of the TGF-β binding integrins. Knockout mice suggest essential roles for integrin-mediated TGF-β activation in vessel and craniofacial morphogenesis during development and in immune homeostasis and the fibrotic wound healing response in the adult. Amplification of integrin-mediated TGF-β activation in fibrotic disorders and data from preclinical models suggest that integrins may therefore represent novel targets for antifibrotic therapies.The multifunctional cytokine transforming growth factor-β (TGF-β) plays major roles in the biology of immune, endothelial, epithelial, and mesenchymal cells during development and adult life in invertebrate and vertebrate species.^1,2 In mammals, these functions are mediated by three isoforms, TGF-β1, 2, and 3, which are each widely expressed.³ All three isoforms interact with the same cell surface receptors (TGFBR2 and ALK5) and signal through the same intracellular signaling pathways, which involve either canonical (ie, SMADs) or noncanonical (ie, MAPK, JUN, PI3K, PP2A, Rho, PAR6) signaling effectors.^4,5 The canonical TGF-β signaling pathway, whereby TGF-β signaling is propagated from the TGF-β receptor apparatus through phosphorylation of cytoplasmic SMAD-2/3, complex formation with SMAD-4, nuclear translocation of the SMAD-2/3/4 complex, and binding to SMAD response elements located in the promoter regions of many genes involved in the fibrogenic response, has been the most intensively studied.⁶ However, despite having similar signaling partners, each isoform serves individual biological functions, perhaps due to differences in binding affinity to TGF-β receptors, activation mechanism, signaling intensity or duration, or spatial and/or temporal distribution.⁷Knockout and conditional deletion models of TGF-β isoforms, receptors, and signaling mediators, as well as function-blocking reagents targeting all TGF-β isoforms, have revealed essential roles for TGF-β in T-cell, cardiac, lung, vascular, and palate development.^{8,9,10,11,12,13,14,15} For instance, mice deficient in TGF-β1 either die in utero owing to defects in yolk sac vasculogenesis or are born and survive into adult life but develop severe multiorgan autoimmunity.¹² Genetic deletion of TGF-β signaling mediators has shown an essential role for Smad2 in early patterning and mesodermal formation,^16,17 and mice lacking Smad3 are viable and fertile, but exhibit limb malformations,¹⁸ immune dysregulation, colitis,¹⁹ colon carcinomas,²⁰ and alveolar enlargement.²¹In adult tissues, the TGF-β pathway is thought to regulate the dynamic interactions among immune, mesenchymal, and epithelial cells to maintain homeostasis in response to environmental stress.²² The normal homeostatic pathways mediated by TGF-β are perturbed in response in chronic repetitive injury. In cases of injury, TGF-β becomes a major profibrogenic cytokine, delaying epithelial wound healing by inhibiting epithelial proliferation and migration and promoting apoptosis and expanding the mesenchymal compartment by inducing fibroblast recruitment, fibroblast contractility, and extracellular matrix deposition.²³ Indeed, intratracheal transfer of adenoviral recombinant TGF-β1 to the rodent lung dramatically increases fibroblast accumulation and expression of type I and type III collagen around airways and in the pulmonary interstitium,^24,25 and neutralizing anti-TGF-β antibodies can block experimental bleomycin or radiation-induced pulmonary fibrosis.^26,27 Increased activity of the TGF-β pathway has also been implicated in fibrotic lung disease, glomerulosclerosis, and restenosis of cardiac vessels.^23,28,29,30 Most TGF-β-mediated pathological changes appear to be attributed to the TGF-β1 isoform.³¹The complexity of TGF-β1 function in humans is illuminated by hereditary disorders with generalized or cell type-specific enhancement or deficiency in either TGF-β1 itself or its signaling effectors. Mutations that increase the activity of the TGF-β pathway lead to defects in bone metabolism (ie, Camurati-Engelmann disease) and in connective tissue (ie, Marfan syndrome), and in aortic aneurysms (ie, Loeys-Dietz syndrome), whereas mutations that lead to decreased activity of the TGF-β pathway correlate with cancer occurrence and prognosis.³² The role of TGF-β as a tumor suppressor in cancer is not straightforward, however, because TGF-β can also enhance tumor growth and metastasis, perhaps through its roles in immune suppression, cell invasion, epithelial-mesenchymal transition, or angiogenesis.^19,33,34,35Despite the multiple essential functions of TGF-β, a single dose or short-term administration of a pan-TGF-β neutralizing antibody is reportedly well tolerated at doses that inhibit organ fibrosis or experimental carcinoma cell growth and metastasis, with no reported side effects in adult mice and rats. This treatment has shown therapeutic efficacy in inhibiting experimental fibrosis.^{27,28,36,37,38,39,40} Because of these promising results, single-dose phase I/II clinical trials using neutralizing pan-TGF-β antibodies have been performed or are ongoing for metastatic renal cell carcinoma, melanoma, focal segmental glomerulosclerosis, and idiopathic pulmonary fibrosis (Genzyme Corporation, http://www.genzymeclinicalresearch.com, last accessed August 27, 2009). However, it is likely that long-term global inhibition of TGF-β will have undesirable side effects, because targeted deletion of TGF-β signaling in various cell types may lead to accelerated atherosclerosis, autoimmunity, or carcinoma development.^9,12,41 Clearly, careful targeting of the TGF-β pathway to minimize systemic effects is a highly desirable goal.     

A Key Role for the Integrin α2β1 in Experimental and Developmental Angiogenesis

The α2β1 integrin receptor plays a key role in angiogenesis. Here we investigated the effects of small molecule inhibitors (SMIs) designed to disrupt integrin α2 I or β1 I-like domain function on angiogenesis. In unchallenged endothelial cells, fibrillar collagen induced robust capillary morphogenesis. In contrast, tube formation was significantly reduced by SMI496, a β1 I-like domain inhibitor and by function-blocking anti-α2β1 but not -α1β1 antibodies. Endothelial cells bound fluorescein-labeled collagen I fibrils, an interaction specifically inhibited by SMI496. Moreover, SMI496 caused cell retraction and cytoskeletal collapse of endothelial cells as well as delayed endothelial cell wound healing. SMI activities were examined in vivo by supplementing the growth medium of zebrafish embryos expressing green fluorescent protein under the control of the vascular endothelial growth factor receptor-2 promoter. SMI496, but not a control compound, interfered with angiogenesis in vivo by reversibly inhibiting sprouting from the axial vessels. We further characterized zebrafish α2 integrin and discovered that this integrin is highly conserved, especially the I domain. Notably, a similar vascular phenotype was induced by morpholino-mediated knockdown of the integrin α2 subunit. By live videomicroscopy, we confirmed that the vessels were largely nonfunctional in the absence of α2β1 integrin. Collectively, our results provide strong biochemical and genetic evidence of a central role for α2β1 integrin in experimental and developmental angiogenesis.Angiogenesis is the formation of new capillaries from pre-existing blood vessels and is essential for human development, wound healing, and tissue regeneration.¹ Angiogenesis is dependent on interactions of endothelial cells with growth factors and extracellular matrix components.^2,3 Endothelial cell-collagen interactions are thought to play a role in angiogenesis in vivo and in vitro and require the function of the α1β1 and α2β1 integrins,³ two receptors known to cross talk.⁴ Thus, vascular endothelial growth factor (VEGF)-induced angiogenesis in Matrigel plugs implanted in mice is markedly inhibited by anti-α1β1 and -α2β1 integrin antibodies.^5,6 Studies using various collagen-induced angiogenesis assays also suggest a critical role for endothelial cell α2β1 integrin^2,7,8 binding to the GFPGER_502–507 sequence of the collagen triple helix.⁹ Consistent with these findings, endorepellin, a potent anti-angiogenic molecule derived from the C terminus of perlecan^10,11 disrupts α2β1 integrin function,^{12,13,14,15,16} and some of the affected gene products have been associated with the integrin-mediated angiogenesis.¹⁷ Endothelial cell-collagen interactions may also contribute to tumor-associated angiogenesis.¹⁸ For example, gene products up-regulated in tumor-associated endothelial cells include types I, III, and VI collagens,¹⁹ and tumor-associated angiogenesis is sensitive to endorepellin treatment.^15,20,21Interestingly, α2β1 integrin-null mice show no overt alteration in either vasculogenesis or angiogenesis but display only a mild platelet dysfunction phenotype and altered branching morphogenesis of the mammary glands.^22,23 This observation suggests that in mammals, there is functional compensation during development, but that α2β1 integrin might be required for postnatal angiogenesis. Indeed, when adult α2β1-null mice are experimentally challenged, they show an enhanced angiogenic response during wound healing²⁴ and tumor xenograft development.^15,25The α1β1 and α2β1 integrins include inserted domains (I domains) in their α subunits that mediate ligand binding.^26,27 The α2 I domain is composed of a Rossman fold and a metal ion coordination site (MIDAS), proposed to ligate the GFPGER_502–507 sequence of collagen, thereby inducing receptor activation.^26,28 Other integrin domains may also play a role in ligand binding and receptor activation. For example, the β1 I-like domain seems to allosterically modulate collagen ligation by the α2 I domain, and, intracellularly, the cytoplasmic sequence of the α2 subunit functions as a hinge, locking the receptor in an inactive conformation, and membrane-soluble peptide mimetics of this sequence were shown to promote α2β1 receptor activation.²⁹ Recently, a family of small molecule inhibitors (SMIs)² targeting the function of the α2β1 integrin were designed.³⁰ Specifically, inhibitors of α2β1 integrin function were prepared using modular synthesis, enabling substitutions of arylamide scaffold backbones with various functional groups, creating SMIs targeted to the I domain or the intact integrin.^30,31,32 In this study, we tested the activities of a group of SMIs on endothelial cell-collagen interactions and angiogenesis in vitro and in vivo. We provide evidence that SMI496, which binds between the I domains of β1 and α2 subunits,³² interferes with α2β1 integrin activity on endothelial cells both in vitro and in vivo, suggesting a potential therapeutic modality to interfere with angiogenesis. Moreover, interference with α2 integrin expression in embryonic zebrafish caused a vascular phenotype characterized by abnormal angiogenesis.     

Lysophosphatidic Acid Induces αvβ6 Integrin-Mediated TGF-β Activation via the LPA2 Receptor and the Small G Protein Gαq

Activation of latent transforming growth factor β (TGF-β) by αvβ6 integrin is critical in the pathogenesis of lung injury and fibrosis. We have previously demonstrated that the stimulation of protease activated receptor 1 promotes αvβ6 integrin-mediated TGF-β activation via RhoA, which is known to modulate cell contraction. However, whether other G protein-coupled receptors can also induce αvβ6 integrin-mediated TGF-β activation is unknown; in addition, the αvβ6 integrin signaling pathway has not yet been fully characterized. In this study, we show that lysophosphatidic acid (LPA) induces αvβ6-mediated TGF-β activation in human epithelial cells via both RhoA and Rho kinase. Furthermore, we demonstrate that LPA-induced αvβ6 integrin-mediated TGF-β activity is mediated via the LPA2 receptor, which signals via Gα_q. Finally, we show that the expression levels of both the LPA2 receptor and αvβ6 integrin are up-regulated and are spatially and temporally associated following bleomycin-induced lung injury. Furthermore, both the LPA2 receptor and αvβ6 integrin are up-regulated in the overlying epithelial areas of fibrosis in patients with usual interstitial pneumonia. These studies demonstrate that LPA induces αvβ6 integrin-mediated TGF-β activation in epithelial cells via LPA2, Gα_q, RhoA, and Rho kinase, and that this pathway might be clinically relevant to the development of lung injury and fibrosis.Transforming growth factor (TGF)-β includes a pleiotropic group of cytokines that exist in three mammalian isoforms (TGF-β1, -β2, and -β3) that are all secreted as latent complexes. This latent complex needs to be activated for TGF-β family members to exert their biological effect. The small latent complex contains the latency associated peptide (LAP), which, in TGF-β1 and TGF-β3, contains an arginine-glycine-aspartate (RGD) motif. This RGD motif can bind integrins, facilitating TGF-β activation. The LAP of TGF-β2 does not contain an RGD motif and no role for integrin mediated TGF-β2 activation has been described. TGF-β1 exerts profound effects on matrix deposition and is a central mediator of lung injury and fibrosis. There are several mechanisms by which TGF-β1 may be activated, including extremes of heat, oxidation, proteolytic cleavage, deglycosylation, and activation by thrombospondin-1.^{1,2,3,4,5,6,7,8} In vivo, activation by integrins appears to play a major role in activating TGF-β1 during development⁹ and in various disease models.^{10,11,12,13,14}Integrins are heterodimeric transmembrane proteins made up of α and β subunits. Six, of the 24 currently described integrins are able to bind the RGD motif in the LAP of TGF-β. Four of these integrins (αvβ3, αvβ5, αvβ6, and αvβ8) are thought to be able to activate TGF-β1.^13,14,15 The role of integrin-mediated TGF-β activation in vivo has only been confirmed for the αvβ6 and αvβ8 integrins.^13,14 Mice in which the aspartic acid in the RGD site of TGF-β1 is replaced by glutamic acid, preventing integrin-mediated TGF-β1 activation, completely phenocopy TGF-β1 null mice, highlighting the importance of TGF-β1 interactions with integrins.⁹ Furthermore, activation of TGF-β1 by the epithelially restricted αvβ6 integrin is central to the pathogenesis of acute lung injury and pulmonary fibrosis.^12,14Further regulation of TGF-β bioavailability is afforded by interaction of the small latent complex with the latent TGF-β binding proteins (LTBPs). There are four LTBPs (1, 2, 3, and 4) that belong to the LTBP/fibrillin family of extracellular glycoproteins. Of these, three, LTBP-1, -3, and -4, associate with the small latent complex through covalent attachment with the LAP, forming the large latent complex.¹⁶ The LTBPs are required to ensure correct post-translational modification of the small latent complex,¹⁷ and they target storage of TGF-β in the extracellular matrix by crosslinking the large latent complex to the matrix via the actions of tissue transglutaminase.^18,19 The LTBPs are also likely to determine, at least in part, the specificity of TGF-β activation. LTBPs-1 and -3 can bind all isoforms of TGF-β, whereas LTBP-4 can only bind TGF-β1.^16,20 There is further evidence of the importance of LTBP modulating TGF-β activation from in vivo studies using mice null for various LTBPs. LTBP-1 null mice have reduced TGF-β activity and are protected from hepatic fibrosis.²¹ LTBP-3 null mice have phenotypic features consistent with reduced TGF-β activity in the bones.²² Mice with a gene trap disruption of LTBP-4 show reduced epithelial Smad2 phosphorylation and abnormal cardiopulmonary development and develop colonic tumors similar to those seen in Smad3 null mice.²³Overexpression of the αvβ6 integrin is not sufficient to promote fibrosis and the αvβ6 integrin itself must be activated during injury to promote TGF-β1 activation.^12,24 αvβ6-dependent TGF-β activation also requires an intact actin cytoskeleton¹⁴ and is critically dependent on association of latent complexes with the specific LTBP family member, LTBP-1.²⁵ In cells lacking LTBP-1, αvβ6 cannot activate TGF-β, but this response can be rescued by expression of a short fusion protein composed of the region of LTBP-1 that forms a disulfide bond to TGF-β1 LAP and the region required to cross-link LTBP-1 to the extracellular matrix protein fibronectin.²⁵ These findings are consistent with a model by which the integrin-expressing cell activates latent TGF-β by exerting traction on the tethered latent complex. Such a mechanism received further support from a recent report that myofibroblasts can activate latent TGF-β using the αvβ5 integrin and cell contraction.²⁶ It is thought that cellular injury may induce contractile forces through the cytoskeleton and integrins to the latent TGF-β1 molecule, which is itself tethered by the LTBP-1 to either the cell surface or the extracellular matrix.^25,26 We identified that agonists of the seven-transmembrane domain, G protein-coupled receptor (GPCR), PAR1, can promote αvβ6 integrin-mediated TGF-β activation via RhoA, known to modulate cell contraction, and that this pathway is important in acute lung injury.¹² However, whether other GPCRs are able to contribute to αvβ6 integrin-mediated, TGF-β activation is not known. Furthermore, the G protein involved in mediating injury-induced αvβ6 integrin-mediated, TGF-β activation has not yet been identified.Lysophosphatidic acid (LPA), is a bioactive phospholipid known to mediate contraction in a number of cell types.²⁷ It is released from activated platelets at sites of injury,²⁸ contributing to wound repair. LPA is present in bronchoalveolar lavage fluid and increased during inflammation,²⁹ and can mediate pro-inflammatory effects on several cells types within the lung.^30,31,32 Furthermore, LPA is increased in patients with pulmonary fibrosis.³³ LPA mediates its cellular effects via the LPA class of GPCRs. Currently five subtypes have been identified (LPA1-5), and like PAR1, these receptors couple to the small G proteins Gα_i, Gα_q, and Gα_12/13.^34,35 It has recently been shown that LPA can induce fibroblast chemotaxis through an LPA1-dependent mechanism, and this could be important in the pathogenesis of pulmonary fibrosis.³³The purpose of the present study was to investigate whether LPA can activate TGF-β in epithelial cells via the αvβ6 integrin and to dissect the proximal signaling pathway from the relevant LPA receptor to RhoA. We demonstrate that the LPA2 receptor is the predominant receptor for transducing LPA-induced, αvβ6 integrin-mediated, TGF-β activation and that this pathway is coupled to the G protein, Gα_q. Finally we provide evidence that both the αvβ6 integrin and LPA2 receptor are induced in epithelial cells overlying areas of pulmonary fibrosis in the lungs of mice treated with intratracheal bleomycin and in samples from patients with idiopathic pulmonary fibrosis.     

ICAM-1 Is Necessary for Epithelial Recruitment of γδ T Cells and Efficient Corneal Wound Healing

Wound healing and inflammation are both significantly reduced in mice that lack γδ T cells. Here, the role of epithelial intercellular adhesion molecule-1 (ICAM-1) in γδ T cell migration in corneal wound healing was assessed. Wild-type mice had an approximate fivefold increase in epithelial γδ T cells at 24 hours after epithelial abrasion. ICAM-1^−/− mice had 50.9% (P < 0.01) fewer γδ T cells resident in unwounded corneal epithelium, which failed to increase in response to epithelial abrasion. Anti-ICAM-1 blocking antibody in wild-type mice reduced epithelial γδ T cells to a number comparable to that of ICAM-1^−/− mice, and mice deficient in lymphocyte function-associated antigen-1 (CD11a/CD18), a principal leukocyte receptor for ICAM-1, exhibited a 48% reduction (P < 0.01) in peak epithelial γδ T cells. Re-epithelialization and epithelial cell division were both significantly reduced (∼50% at 18 hours, P < 0.01) after abrasion in ICAM-1^−/− mice versus wild-type, and at 96 hours, recovery of epithelial thickness was only 66% (P < 0.01) of wild-type. ICAM-1 expression by corneal epithelium in response to epithelial abrasion appears to be critical for accumulation of γδ T cells in the epithelium, and deficiency of ICAM-1 significantly delays wound healing. Since γδ T cells are necessary for efficient epithelial wound healing, ICAM-1 may contribute to wound healing by facilitating γδ T cell migration into the corneal epithelium.Intercellular adhesion molecule-1 (ICAM-1, CD54)¹ is a conserved member of the immunoglobulin supergene family² and is expressed by many cell types in response to stimuli such as cytokines,^3,4 and oxidative and physical stress.^5,6 It has been extensively studied in the context of adhesion and transmigration of leukocytes through endothelium⁷ and epithelium,^8,9 and it also serves as an adhesive ligand for leukocyte-mediated cytotoxic activity.^9,10,11 ICAM-1 is recognized by members of the β2 (CD18) integrin family, especially lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18),¹² and this adhesion is critical to many of the migratory and cytotoxic events in which ICAM-1 participates.^7,10,11 ICAM-1 also functions as a signaling molecule, dependent on its cytoplasmic tail interacting with cytoskeletal elements.⁷ This capability influences functions such as leukocyte transendothelial migration⁷ and vascular permeability.¹³Of importance to the current study is the fact that ICAM-1 can be expressed by corneal epithelial cells and limbal vessel endothelial cells.^{14,15,16,17,18,19,20,21} It appears to be expressed in conditions associated with inflammation, but its role in this context is poorly understood, especially its expression by the epithelial cells. Using a murine model of central corneal epithelial abrasion, we observed ICAM-1 on corneal epithelial cells in the periphery of the cornea, a region not directly injured by the abrasion.¹⁴ Since migration and division of these cells account for wound closure and re-establishment of full thickness epithelium necessary for healing,^22,23 it was of interest to determine whether ICAM-1 is necessary for these processes. To this end we studied wound healing in mice that do not express ICAM-1.^24,25As a part of this evaluation, we focused attention on γδ T cells. We observed in earlier studies that epithelial expression of ICAM-1 occurred at a time when γδ T cells increased within the corneal epithelium,^14,26 and that γδ T cell-deficient mice exhibited poor corneal wound healing. Since these leukocytes express LFA-1,²⁷ and LFA-1/ICAM-1 interactions support adhesion of human lymphocytes to human epithelial cells expressing ICAM-1,^20,27 it seemed possible that γδ T cell accumulation in the epithelium after corneal abrasion would be influenced by the absence of ICAM-1.     

Laminin α4-Null Mutant Mice Develop Chronic Kidney Disease with Persistent Overexpression of Platelet-Derived Growth Factor

Each extracellular matrix compartment in the kidney has a unique composition, with regional specificity in the expression of various laminin isoforms. Although null mutations in the majority of laminin chains lead to specific developmental abnormalities in the kidney, Lama4−/− mice have progressive glomerular and tubulointerstitial fibrosis. These mice have a significant increase in expression of platelet-derived growth factor (PDGF)-BB, PDGF-DD, and PDGF receptor β in association with immature glomerular and peritubular capillaries. In addition, mesangial cell exposure to α4-containing laminins, but not other isoforms, results in down-regulation of PDGF receptor mRNA and protein, suggesting a direct effect of LN411/LN421 on vessel maturation. Given the known role of overexpression of PDGF-BB and PDGF-DD on glomerular and tubulointerstitial fibrosis, these data suggest that failure of laminin α4-mediated down-regulation of PDGF activity contributes to the progressive renal lesions in this animal model. Given the recent demonstration that individuals with laminin α4 mutations develop cardiomyopathy, these findings may be relevant to kidney disease in humans.Laminin (LN) is a large, heterotrimeric, cruciform molecule composed of α, β, and γ subunits.¹ Five distinct α (LAMA1-5), 3 β (LAMB1-3), and 3 γ (LAMC1-3) chains^1,2 variably assemble to create distinct isoforms³ that are temporally and spatially regulated, and each conveys a variety of biological functions.^{4,5,6,7,8,9,10,11} The LNα4-containing isoforms, LN411 (α4β1γ1) and LN421 (α4β2γ1), are abundant in microvessels. Studies of LNα4-deficient mutant mice (lama4−/−) reveal that although α4-LNs are not required for blood vessel formation, they play important roles in blood vessel maturation, and in stabilization of vessels that form with injury, inflammation and tumor growth.^7,12,13 In vitro studies indicate that α4LNs directly regulate endothelial cell proliferation and inhibit apoptosis.¹⁴ α4-LNs are produced by endothelial cells in most microvessels; however, endothelial cells in the renal glomerulus do not express LNα4-containing isoforms.¹⁵ Instead, LN411 and LN421 at the endothelial-mesangial interface are produced by the mesangial cells (MCs).¹⁵ Platelet-derived growth factor (PDGF) is the primary growth factor responsible for MC proliferation and migration during glomerulogenesis,¹⁶ and we have shown that PDGF-induced MC migration requires LNα4.¹⁵ This function could not be replaced by LN111 or LN511/521.¹⁵ Together these observations suggested the possibility that deficiency of LNα4 might impair the ability of the kidney microvasculature to mature or be repaired in lama4−/− adult mice, resulting in kidney disease despite normal initial development.Previous reports have documented a spectrum of developmental defects and tissue maintenance defects in lama4−/− mice. Early postal-natal hemorrhage from birth-related trauma to fragile blood vessels occurs in lama4−/− mice; yet, by three-weeks of age, accumulation of LNα5 stabilizes vessels, although they remain dilated.¹² Vessel fragility recurs when new vessels form in response to injury.¹² The heart forms normally, but lama4−/− mice develop cardiomyopathy with time.¹⁷ Neurological dysfunction occurs in lama4−/− mice, through independent defects in organizing presynaptic specializations at neuromuscular synapses,¹⁸ and in the ability of developing Schwann cells to properly sort and myelinate.^19,20 This report details the characteristics of kidney abnormalities, including the development of glomerulosclerosis and tubulointerstitial fibrosis over time in lama4−/− mice.     

Two Different PDGF β-Receptor Cohorts in Human Pericytes Mediate Distinct Biological Endpoints

How activation of a specific growth factor receptor selectively results in either cell proliferation or cytoskeletal reorganization is of central importance to the field of pathophysiology. In this study, we report on a novel mechanism that explains how this process is accomplished. Our current investigation demonstrates that soluble platelet derived growth factor- (PDGF)-BB activates a cohort of PDGF-β receptors primarily confined to the lipid raft component of the cell membrane, specifically caveolae. In contrast, cell-bound PDGF-BB delivered via cell–cell contact results in activation and the subsequent up-regulation of a cohort of PDGF β-receptors primarily confined to the non-lipid raft component of the cell membrane. Individual activation of these two receptor cohorts results in distinct biological endpoints, cytoskeletal reorganization or cell proliferation. Mechanistically, our evidence suggests that PDGF-BB-bearing cells preferentially stimulate the non-lipid raft receptor cohort through interleukin 1β-mediated inhibition of the lipid raft cohort of receptors, leaving the non-raft receptor cohort operational and preferentially stimulated. In human skin injected with PDGF-BB and in tissue reparative processes PDGF β-receptors colocalize with the caveolae/lipid raft marker caveolin-1. In contrast, in human skin injected with PDGF-BB-bearing tumor cells and in colorectal adenocarcinoma, activated PDGF β-receptors do not colocalize with caveolin-1. Thus, growth factor receptors are segregated into specific cell membrane compartments that are preferentially activated through different mechanisms of ligand delivery, resulting in distinct biological endpoints.Lipid rafts are cellular membrane domains that contain high concentrations of cholesterol and sphingolipids. These domains include the flat and related vesicular structures referred to as caveolae. Caveolae, which are formed by the macromolecular oligomerization of the 22-kDa caveolin protein are enriched in a number of vital signal transduction molecules, and contain smaller cohorts of many others.^1,2,3,4,5,6 Furthermore, caveolin itself directly binds and/or regulates the activities of a number of these signaling molecules.¹ With specific regards to the platelet derived growth factor (PDGF) signaling axis, PDGF-induced signaling occurs in caveolae of many mesenchymal cells,^5,6,7,8 and PDGF-receptors are functional in isolated caveolae.⁸ Based on the apparent signaling events occurring in lipid rafts, and the abundance of molecules involved in multiple signaling pathways, it is inferred that lipid rafts are important loci for signal amplification and cross talk between signaling pathways.^1,2,5,6,7,8 Recently emerging evidence shows that lipid rafts also have important specific roles in regulating the activity of cytoskeleton-regulating GTPases, in cytoskeletal organization, in the formation of cell extensions, and in cell motility.⁹The PDGF-B chain contains a retention motif that mediates binding to heparan sulfate proteoglycans on cell surfaces.¹⁰ This affords PDGF-BB-producing cells alternate modes of ligand delivery to PDGF β-receptor bearing cells, through heterotypic cell-to-cell contacts or as a secreted soluble ligand.^11,12 In mesenchymal cell–tumor cell co-cultures, activation of PDGF β-receptors is a consequence of cell–cell contacts, and is not accomplished via soluble PDGF-BB.¹³ The biological consequences of cell–cell versus secreted ligand remain unknown. Given the central role of PDGF β-receptor activation in pericyte biology during embryogenesis and reactive conditions in the adult organism,^{14,15,16,17,18,19,20} we chose to investigate the role of caveolae in PDGF β-receptor signaling in primary human pericytes. Activation of PDGF β-receptors in mesenchymal cells leads to several biological endpoints, eg, proliferation and reorganization of the actin cytoskeleton.²⁰ How cells are able to orchestrate signal transduction events leading to different biological endpoints, in response to stimulation by a specific ligand, is not known. Here we demonstrate one mechanism by which context-specific ligand stimulation of a growth factor receptor results in distinct biological endpoints.     

Toll-Like Receptor 3 Ligand Dampens Liver Inflammation by Stimulating Vα14 Invariant Natural Killer T Cells to Negatively Regulate γδT Cells

Vα14 invariant natural killer T (Vα14iNKT) cells are at the interface between the innate and adaptive immune responses and are thus critical for providing full engagement of host defense. We investigated the role of polyriboinosinic:polycytidylic acid (poly I:C), a replication-competent viral double-stranded RNA mimic and a specific agonist that recognizes the cellular sensor Toll-like receptor 3 (TLR3), in regulating Vα14iNKT cell activation. We established for the first time that hepatic Vα14iNKT cells up-regulate TLR3 extracellularly after poly I:C treatment. Notably, activation of TLR3-expressing hepatic Vα14iNKT cells by a TLR3 ligand was suppressed by TLR3 deficiency. Our studies also revealed that Vα14iNKT cell activation in response to poly I:C administration uniquely suppressed the accumulation and activation of intrahepatic γδT cells (but not natural killer cells) by inducing apoptosis. Furthermore, we established that activated hepatic Vα14iNKT cells (via cytokines and possibly reactive oxygen species) influenced the frequency and absolute number of intrahepatic γδT cells, as evidenced by increased hepatic γδT cell accumulation in Vα14iNKT cell-deficient mice after poly I:C treatment relative to wild-type mice. Thus, hepatic Vα14iNKT cells and intrahepatic γδT cells are functionally linked on application of TLR3 agonist. Overall, our results demonstrate a novel and previously unrecognized anti-inflammatory role for activated hepatic Vα14iNKT cells in negatively regulating intrahepatic γδT cell accumulation (probably through TLR3 signaling) and thereby preventing potentially harmful activation of intrahepatic γδT cells.Vα14 invariant natural killer T (Vα14iNKT) cells are thymic-derived innate murine T lymphocytes with significant immunoregulatory effects in cardiovascular, infectious, and autoimmune diseases as well as in tumors.^{1,2,3,4,5,6,7,8,9,10} In contrast to conventional T cells, which recognize peptide antigens presented by major histocompatability class I and II molecules, Vα14iNKT cells respond to glycolipid antigen presented by CD1d expressed on antigen-presenting cells.^11,12 In the last decade, several potential mechanisms underlying Vα14iNKT cell activation during immune responses have been revealed. Vα14iNKT cells are activated by lipids presented by CD1d.^5,13 The established dogma is that the lipid tail of glycolipid antigen (including α-galactosylceramide or exogenous antigens from pathogens) is buried in CD1d, whereas the sugar head group of glycolipid antigen protrudes out of the CD1d to activate the T-cell receptor (TCR) α on the Vα14iNKT cell.^2,13,14 After activation, Vα14iNKT cells exert multiple effects including the production of several cytokines (such as interferon [IFN]-γ, interleukin [IL]-4, and tumor necrosis factor [TNF]-α), chemokines (regulated on activation normal T cell expressed and secreted/CCL5, monocyte chemotactic protein-1/CCL2, and macrophage inflammatory protein-1α/CCL3)^15,16,17,18 and cytotoxic proteins (such as tumor necrosis factor-related apoptosis-inducing ligand and Fas/Fas ligand).^13,19 Through these mediators, activated Vα14iNKT cells can interact with and transactivate other immune cells.^15,20,21 Thus, Vα14iNKT cells act as a “bridge” between the innate and adaptive immune systems.Vα14iNKT cells are also activated by a TCR-independent mechanism involving Toll like receptors (TLRs). TLRs are pathogen recognition receptors that identify molecular patterns of components specific to microbes and play a critical role in initiating the innate immune response to microbes.²² To date, more than 10 TLRs have been reported in humans and mice, and each recognizes different microbial components.²² TLRs are located on the plasma membrane and in endosomal compartments of cells.²² Among the TLRs, TLR2, TLR4, and TLR5 recognize the bacterial signals peptidoglycan, lipopolysaccharide, and flagellin, respectively,²² whereas TLR3, TLR7, TLR8, and TLR9 play fundamental roles in detecting viral signals.^23,24,25,26 An additional mechanism for activation of Vα14iNKT cells (in the absence of foreign antigen for their TCRs) is by IL-12 and/or IL-18 derived from antigen-presenting cells that have been activated via a TLR (4,7,8,9)-dependent pathway.^13,14,27,28 The precise pathway of activation may depend on the pathogen. For example, TLR4 traditionally recognizes the bacterial signal lipopolysaccharide,²² whereas TLR7, TLR8, and TLR9 all sense viral signals.^23,24,25,26 The potential contribution of the viral sensor TLR3 to Vα14iNKT cell activation has not yet been determined. Poly I:C is the specific TLR3 agonist and a replication-competent viral double-stranded RNA (dsRNA) mimic.^23,29 dsRNA is a structure found in the genome of some viruses and is produced as a replication intermediate by viruses.^23,29,30 Therefore, poly I:C is routinely used in experimental studies to assess the functional activity of TLR3 during immune responses.^23,29,30,31In the present study, we evaluated the potential role of TLR3 in promoting Vα14iNKT cell activation by examining the response of hepatic Vα14iNKT cells after treatment with the TLR3 ligand, poly I:C. In addition, we assessed the functional consequences of Vα14iNKT cell activation on the hepatic innate immune response after poly I:C treatment. We demonstrate that a functional consequence of hepatic Vα14iNKT cell activation in response to poly I:C administration is the subsequent induction of apoptotic death of hepatic γδT cells. Overall, our findings demonstrate a novel role for activated hepatic Vα14iNKT cells in negatively regulating the recruitment, activation, and potentially harmful effector function(s) of intrahepatic γδT cells on application of the TLR3 ligand, poly I:C.     

A Protein Kinase Cδ-Dependent Protein Kinase D Pathway Modulates ERK1/2 and JNK1/2 Phosphorylation and Bim-Associated Apoptosis by Asbestos

Inhalation of asbestos and oxidant-generating pollutants causes injury and compensatory proliferation of lung epithelium, but the signaling mechanisms that lead to these responses are unclear. We hypothesized that a protein kinase (PK)Cδ-dependent PKD pathway was able to regulate downstream mitogen-activated protein kinases, affecting pro- and anti-apoptotic responses to asbestos. Elevated levels of phosphorylated PKD (p-PKD) were observed in distal bronchiolar epithelial cells of mice inhaling asbestos. In contrast, PKCδ−/− mice showed significantly lower levels of p-PKD in lung homogenates and in situ after asbestos inhalation. In a murine lung epithelial cell line, asbestos caused significant increases in the phosphorylation of PKCδ-dependent PKD, ERK1/2, and JNK1/2/c-Jun that occurred with decreases in the BH3-only pro-apoptotic protein, Bim. Silencing of PKCδ, PKD, and use of small molecule inhibitors linked the ERK1/2 pathway to the prevention of Bim-associated apoptosis as well as the JNK1/2/c-Jun pathway to the induction of apoptosis. Our studies are the first to show that asbestos induces PKD phosphorylation in lung epithelial cells both in vivo and in vitro. PKCδ-dependent PKD phosphorylation by asbestos is causally linked to a cellular pathway that involves the phosphorylation of both ERK1/2 and JNK1/2, which play opposing roles in the apoptotic response induced by asbestos.Asbestos is a group of naturally occurring mineral fibers that are linked to the development of lung cancer, mesothelioma, and pleural and pulmonary fibrosis, ie, asbestosis.^1,2 The mechanisms leading to asbestos-related diseases are still unclear, but oxidative stress due to phagocytosis of longer fibers, iron-driven generation of oxidants from fiber surfaces, and depletion of cellular antioxidants are linked to cell injury and inflammation.^3,4,5,6Bronchiolar and alveolar type II epithelial cells, which first encounter asbestos fibers after inhalation, are key cell types in asbestos-associated inflammation and fibroproliferation.² Initial cell reactions to asbestos include epithelial cell injury, ie, apoptosis and necrosis,^5,6 which may lead to compensatory cell proliferation^7,8 and the production of inflammatory and fibrogenic cytokines.^8,9,10 Asbestos-induced signaling mechanisms governing these cell responses appear to involve a broad variety of cascades including the mitogen-activated protein kinases (MAPK),^3,7,11,12 nuclear factor-κB (NF-κB),^9,13,14 and the protein kinase (PK)C^10,12,15,16 and A families.¹⁷A critical signaling protein involved in asbestos signaling is PKCδ, which is known to be activated in bronchiolar and alveolar epithelial cells in vivo and in vitro^10,12,16 via increased formation of diacylglycerol.¹⁸ We have shown that PKCδ governs apoptosis via an oxidant-dependent mitochondrial pathway after exposure of lung epithelial cells to asbestos fibers.¹⁶ Recent studies comparing PKCδ +/+ and PKCδ −/− mice also reveal an important role of PKCδ in metalloproteinase expression as well as cytokine production in vitro and in vivo.^10,15 A variety of other studies also link PKCδ to either pro-apoptotic or anti-apoptotic events depending on the stimulus and cell type.^19,20In this study, we focused on PKD as a potential link between PKCδ, activation of MAPKs and downstream repercussions such as expression of fos/jun proto-oncogenes and apoptosis in asbestos-exposed lung epithelium. PKD is a serine/threonine protein kinase classified as a subfamily of the Ca²⁺/calmodulin-dependent kinase superfamily.²¹ PKD1, which includes mouse PKD and its human homolog PKCμ, is the most extensively studied PKD.²² The other two members of this family include PKD2²³ and PKD3, (originally PKCν).²⁴ Conserved regions of PKDs include a phosphorylation-dependent catalytic domain, a pleckstrin-homology domain that inhibits the catalytic activity, and cysteine-rich motifs that recruit PKD to the plasma membrane. PKCδ is proposed to interact with the pleckstrin-homology domain of PKD, transphosphorylating its activation loop at Ser744 and Ser748, and leading to PKD activation.²⁵ In addition, PKD can be activated through the Src-Abl pathway by tyrosine phosphorylation of Tyr463 (T463) in the pleckstrin-homology domain after oxidative stress,²⁶ as well as by caspase-mediated proteolytic cleavage 27 and by bone morphogenetic protein 2.²⁸ Downstream targets of PKD signaling include several important signaling molecules such as ERK1/2, JNK1/2, and NF-κB,^21,26,29,30 but how these affect functional ramifications of carcinogens, such as asbestos, are unclear.The BH3-only protein, Bim, is a pro-apoptotic member of the Bcl-2 family that links stress-induced signals to the core apoptotic machinery.^31,32 There are three different splice variants of the Bim gene encoding short, long, and extra-long Bim proteins (BimS, BimL, and BimEL).³³ BimS-induced apoptosis requires mitochondrial localization but not interaction with anti-apoptosis proteins,³⁴ whereas BimL is bound to microtubules and is less cytotoxic.³⁵ Disruption of BimL binding to microtubules via JNK-dependent phosphorylation can cause its redistribution to the mitochondria and induction of pro-apoptotic machinery.³⁶ BimEL is post-translationally regulated by ERK1/2, which promotes its phosphorylation and rapid dissociation from Mcl-1 and Bcl-x(L)³⁷ and proteasomal degradation.³⁸We reveal here that PKD is involved in multiple signaling events after asbestos inhalation and in vitro. Specifically, PKD is a downstream effector of PKCδ and modulates phosphorylation of both ERK1/2 and JNK1/2 in lung epithelial cells after asbestos exposure. Our data also suggest that PKD inhibits apoptosis through an ERK1/2-mediated destabilization of the pro-apoptotic BH3-only protein, BimEL. The fact that PKD is an important signaling molecule in MAPK signaling and survival after cell injury by asbestos may have important therapeutic implications in asbestos-related diseases.     

Differential Interferon Responses Enhance Viral Epitope Generation by Myocardial Immunoproteasomes in Murine Enterovirus Myocarditis

Murine models of coxsackievirus B3 (CVB3)-induced myocarditis mimic the divergent human disease course of cardiotropic viral infection, with host-specific outcomes ranging from complete recovery in resistant mice to chronic disease in susceptible hosts. To identify susceptibility factors that modulate the course of viral myocarditis, we show that type-I interferon (IFN) responses are considerably impaired in acute CVB3-induced myocarditis in susceptible mice, which have been linked to immunoproteasome (IP) formation. Here we report that in concurrence with distinctive type-I IFN kinetics, myocardial IP formation peaked early after infection in resistant mice and was postponed with maximum IP expression concomitant to massive inflammation and predominant type-II IFN responses in susceptible mice. IP activity is linked to a strong enhancement of antigenic viral peptide presentation. To investigate the impact of myocardial IPs in CVB3-induced myocarditis, we identified two novel CVB3 T cell epitopes, virus capsid protein 2 [285-293] and polymerase 3D [2170-2177]. Analysis of myocardial IPs in CVB3-induced myocarditis revealed that myocardial IP expression resulted in efficient epitope generation. As opposed to the susceptible host, myocardial IP expression at early stages of disease corresponded to enhanced CVB3 epitope generation in the hearts of resistant mice. We propose that this process may precondition the infected heart for adaptive immune responses. In conclusion, type-I IFN-induced myocardial IP activity at early stages coincides with less severe disease manifestation in CVB3-induced myocarditis.Myocarditis is often induced by cardiotropic viruses: in about 20% of patients, viral myocarditis leads to its sequela dilated cardiomyopathy, which is linked to chronic inflammation and persistence of cardiotropic viruses.^1,2,3,4 Dilated cardiomyopathy is the most common cause of heart failure in young patients and appears to be a major cause of sudden unexpected death in this cohort. Enteroviruses, including group-B coxsackieviruses, have been linked to the development of myocarditis and dilated cardiomyopathy associated with adverse prognosis.^5,6 Well-established murine models of coxsackievirus B3 (CVB3) myocarditis mimic the human disease progress and are valuable in delineating the underlying mechanisms that determine the divergent courses of myocarditis^7,8,9,10: resistant C57BL/6 mice eliminate the virus following mild acute myocarditis; no chronic inflammation is detected. In contrast, major histocompatibility complex (MHC)-matched A.BY/SnJ mice develop severe acute infection and ongoing chronic myocarditis, thus conferring susceptibility to chronic disease.^7,9Host responses to viral infection trigger the release of interferons (IFNs). IFNs of the α/β subtype are assigned to type I IFNs, whereas IFN-γ is the only type II IFN. IFNs exert numerous antiviral effects in innate and adaptive immunity.¹¹ Although type I IFN-receptor-deficiency was not associated with a dramatic effect on early viral replication in the heart, type I IFN signaling was found to be essential for the prevention of early death due to CVB3-infection.¹² The extraordinary impact of type I IFNs was substantiated in a recent study illustrating acute fulminant infection and chronic disease progression in IFN-β deficient mice.¹³ Deletion of type II IFN receptors was not associated with enhanced mortality in CVB3-infection.¹² IFN-γ responses were shown to be protective in cellular immunity in CVB3-infection.⁹ In addition, expression of IFN-γ conferred protection in enterovirus myocarditis, which may be linked to the activation of nitric oxide-mediated antiviral activity of macrophages.^14,15 Thus, both type I and type II IFN are active in CVB3- myocarditis.One downstream effect of IFN signaling is the induction of immunoproteasome (IP) formation in the target organ of the immune response. Particularly IFN-γ was shown to induce IP expression.^16,17,18 Efficient generation of viral epitopes that stimulate CD8⁺ T cells strongly relies on host-cell IP and, in addition, protein degradation by proteasomes is also essential in the regulation of inflammatory and stress responses, cell cyclus, and apoptosis control.¹⁹ The 20S proteasome as the catalytic core of the proteasome resembles a cylinder-shaped structure of stacked heptameric rings formed by either α or β subunits. The proteolytic function of the so-called standard proteasome is restricted to the β1, β2, and β5 subunit.²⁰ Three alternative catalytic subunits, the so-called immunosubunits β1i, β2i, and β5i, which are incorporated into 20S proteasomes, thus forming IP with altered catalytic characteristics, are expressed on cytokine stimulation.^21,22 It is highly notable that IP activity is linked to a strong enhancement of antigenic viral peptide presentation.^{23,24,25,26,27}Cardiac proteasomes contribute to the modulation of cardiac function in health and disease.²⁸ However, apart from the reported observation that IPs are expressed in the myocardium in acute CVB3 myocarditis, their functional impact has not been studied so far.¹⁰ The present study focuses on IFN-induced myocardial IP activity in CVB3 myocarditis.     

Hyperhomocysteinemia Increases β-Amyloid by Enhancing Expression of γ-Secretase and Phosphorylation of Amyloid Precursor Protein in Rat Brain

Hyperhomocysteinemia and β-amyloid (Aβ) overproduction are critical etiological and pathological factors in Alzheimer disease, respectively; however, the intrinsic link between them is still missing. Here, we found that Aβ levels increased and amyloid precursor protein (APP) levels simultaneously decreased in hyperhomocysteinemic rats after a 2-week induction by vena caudalis injection of homocysteine. Concurrently, both the mRNA and protein levels of presenilin-1, a component of γ-secretase, were elevated, whereas the expression levels of β-secretase and presenilin-2 were not altered. We also observed that levels of phosphorylated APP at threonine-668, a crucial site facilitating the amyloidogenic cleavage of APP, increased in rats with hyperhomocysteinemia, although the phosphorylation per se did not increase the binding capacity of pT668-APP to the secretases. The enhanced phosphorylation of APP in these rats was not relevant to either c-Jun N-terminal kinase or cyclin-dependent kinase-5. A prominent spatial memory deficit was detected in rats with hyperhomocysteinemia. Simultaneous supplementation of folate and vitamin-B12 attenuated the hyperhomocysteinemia-induced abnormal processing of APP and improved memory. Our data revealed that hyperhomocysteinemia could increase Aβ production through the enhanced expression of γ-secretase and APP phosphorylation, causing memory deficits that could be rescued by folate and vitamin-B12 treatment in these rats. It is suggested that hyperhomocysteinemia may serve as an upstream factor for increased Aβ production as seen in patients with Alzheimer disease.Alzheimer’s disease (AD) is a progressive neurological disorder characterized histopathologically by the formation of numerous senile plaques and neurofibrillary tangles. The senile plaques are mainly composed of amyloid-β (Aβ), surrounded by dystrophic neuritis.¹ Aβ is generated by the consecutive cleavage of amyloid precursor protein (APP) by two proteases, ie, β-secretase (BACE-1) and γ-secretase (presenilin, PS-1/PS-2).^2,3 In the amyloidogenic pathway, cleavage of APP by β-secretase generates an N-terminal soluble fragment (sAPPβ) and beta C-terminal fragment that is sequentially cleaved by γ-secretase to produce the Aβ peptides.^4,5,6 Similar to many other toxic insults, Aβ promotes cell death by oxidative damage,^7,8 influencing calcium homeostasis,⁹ activating caspases,¹⁰ stimulating protein phosphorylation,¹¹ and causing mitochondrial abnormalities.¹² In addition, Aβ fibrils specifically induce neuron dystrophy.^13,14 In the cultured rats’ cortical neurons, overexpression of APP induces apoptosis and this apoptosis can be intercepted by γ-secretase inhibitor.¹⁵ In transgenic mouse models, Aβ aggregation induces dysfunction of neurites, tau pathology, and neuron death, and Aβ can also damage DNA.¹⁶ When APP is overexpressed or abnormally cleaved, Aβ forms toxic oligomers that aggregate into amyloid plaques and are associated with age-related memory impairment.¹⁷It is well known that gene mutation of APP and PS-1 is causative for the increased Aβ production in hereditary AD.¹⁸ However, the mechanism leading to the Aβ overproduction in the majority sporadic AD patients is unclear. APP is a phosphoprotein, which have a large N-terminal extracellular domain and a short intracellular C-terminal domain that can be phosphorylated by various protein kinases with well-defined phosphorylation sites.¹⁹ Notably, phosphorylation of APP at Thr668 facilitates the amyloidogenic cleavage and the phosphorylated APP is elevated in AD brain.^20,21Epidemiology and clinical investigations have demonstrated that the elevated plasma homocysteine (Hcy) and the occurrence of AD are positively correlated, and thus hyperhomocysteinemia has been proposed to be a strong and independent risk factor of AD.^{22,23,24,25,26} Hcy is catabolized through the folate and vitamin (vit) B12-dependent remethylation cycle, which provides methyl-group for a number of metabolic steps.²⁷ High Hcy suppresses the cellular levels of S-adenosylmethionine and S-adenosylhomocysteine, and thus inhibits the activity of methyltransferases, which in turn interrupts the methylation of some functional proteins and genes.²⁸ Recently, we have reported that hyperhomocysteinemia can increase prominently the plasma Hcy level and thus induce tau hyperphosphorylation.²⁹ In a hyperhomocysteinemic AD transgenic mouse model, an increased Aβ level in the brain was observed,³⁰ and Hcy could interrupt DNA repair in hippocampal neurons and make the neurons more vulnerable to the amyloid toxicity.^31,32,33 Until now, the effects of hyperhomocysteinemia on Aβ production in normal gene background and the underlying mechanisms leading to Aβ overproduction have not been reported, and it is also not known whether the induced hyperhomocysteinemia in adulthood affects the memory of the rats.In the present study, we produced a hyperhomocysteinemia model in adult rats by injecting Hcy through vena caudalis and investigated the role of hyperhomocysteinemia in Aβ production and the related mechanisms, and as well as the effects on the memory ability of the rats. We found that hyperhomocysteinemia could increase remarkably the Aβ level with concurrent overexpression of PS-1 and hyperphosphorylation of APP at Thr-688, and it also led to spatial memory deficits of the rats. Simultaneous supplementation of folate and vit-B12 could attenuate the hyperhomocysteinemia-induced abnormal APP processing and the memory impairments of the rats.