In this study, we demonstrate that protein kinase C (PKC) activators, including phorbol-12-myristate-13-acetate (PMA), 1,2-dioctanoyl-sn-glycerol (DOG), and platelet-derived growth factor α are potent inducers of angiopoietin-like protein 4 (ANGPTL4) expression in several normal lung cell types and carcinoma cell lines. In human airway smooth muscle (HASM) cells induction of ANGPTL4 expression is observed as early as 2 h after the addition of PMA. PMA also increases the level of ANGPTL4 protein released in the medium. PKC inhibitors Ro31-8820 and Gö6983 greatly inhibit the induction of ANGPTL4 mRNA by PMA suggesting that this up-regulation involves activation of PKC. Knockdown of several PKCs by corresponding siRNAs suggest a role for PKCα. PMA does not activate MAPK p38 and p38 inhibitors have little effect on the induction of ANGPTL4 indicating that p38 is not involved in the regulation of ANGPTL4 by PMA. In contrast, treatment of HASM by PMA induces phosphorylation and activation of Ra, MEK1/2, ERK1/2, JNK, Elk-1, and c-Jun. The Ras inhibitor manumycin A, the MEK1/2 inhibitor U0126, and the JNK inhibitor SP600125, greatly reduce the increase in ANGPTL4 expression by PMA. Knockdown of MEK1/2 and JNK1/2 expression by corresponding siRNAs inhibits the induction of ANGPTL4. Our observations suggest that the induction of ANGPTL4 by PMA in HASM involves the activation of PKC, ERK, and JNK pathways. This induction may play a role in tissue remodeling during lung injury and be implicated in several lung pathologies.

The retinoid-related orphan receptors (ROR) comprise a distinct subfamily of nuclear receptors with the capacity to act as both repressors and activators of transcription. RORγ, the most recently identified member of the ROR family, has been shown to be important for the development of normal lymphocyte compartments as well as organogenesis of some lymphoid organs. In this report, we examine the capacity of RORγ-deficient mice to develop an adaptive immune response to Ag using OVA-induced inflammation in mice as a model for allergic airway disease. In sham-treated mice lacking RORγ, low-grade pulmonary inflammation was observed and characterized by the perivascular accumulation of B and T lymphocytes, increased numbers of inflammatory cells in the lung lavage fluid, and polyclonal Ig activation. Following sensitization and challenge, the capacity of these animals to develop the allergic phenotype was severely impaired as evidenced by attenuated eosinophilic pulmonary inflammation, reduced numbers of CD4+ lymphocytes, and lower Th2 cytokines/chemokine protein and mRNA expression in the lungs. IFN-γ and IL-10 production was markedly greater in splenocytes from RORγ-deficient mice following in vitro restimulation with OVA compared with wild-type splenocytes, and a shift toward a Th1 immune response was observed in sensitized/challenged RORγ-deficient animals in vivo. These data reveal a critical role for RORγ in the regulation of Ig production and Th1/Th2 balance in adaptive immunity.

The retinoid-related orphan receptors (ROR) comprise a distinct subfamily of nuclear receptors with the capacity to act as both repressors and activators of transcription. RORgamma, the most recently identified member of the ROR family, has been shown to be important for the development of normal lymphocyte compartments as well as organogenesis of some lymphoid organs. In this report, we examine the capacity of RORgamma-deficient mice to develop an adaptive immune response to Ag using OVA-induced inflammation in mice as a model for allergic airway disease. In sham-treated mice lacking RORgamma, low-grade pulmonary inflammation was observed and characterized by the perivascular accumulation of B and T lymphocytes, increased numbers of inflammatory cells in the lung lavage fluid, and polyclonal Ig activation. Following sensitization and challenge, the capacity of these animals to develop the allergic phenotype was severely impaired as evidenced by attenuated eosinophilic pulmonary inflammation, reduced numbers of CD4+ lymphocytes, and lower Th2 cytokines/chemokine protein and mRNA expression in the lungs. IFN-gamma and IL-10 production was markedly greater in splenocytes from RORgamma-deficient mice following in vitro restimulation with OVA compared with wild-type splenocytes, and a shift toward a Th1 immune response was observed in sensitized/challenged RORgamma-deficient animals in vivo. These data reveal a critical role for RORgamma in the regulation of Ig production and Th1/Th2 balance in adaptive immunity.

Rationale: Nuclear receptors play a critical role in the regulation of inflammation, thus representing attractive targets for the treatment of asthma.Objective: In this study, we assess the potential regulatory function of retinoid-related orphan receptor alpha (ROR alpha) in the adaptive immune response using ovalbumin (OVA)-induced airway inflammation as a model.Methods: Allergen-induced inflammation was compared between wild-type (WT) and staggerer (ROR alpha(sg/sg)) mice, a natural mutant strain that is deficient in ROR alpha expression.Measurements and Main Results: Despite robust increases in OVA-specific IgE, ROR alpha(sg/sg) mice developed significantly less pulmonary inflammation, mucous cell hyperplasia, and eosinophilia compared with similarly treated WT animals. Induction of Th2 cytokines, including interleukin (IL)-4, IL-5, and IL-13, was also significantly less in ROR alpha(sg/sg) mice. Microarray analysis using lung RNA showed increased expression of many genes, previously implicated in inflammation, in OVA-treated WT mice. These include mucin Muc5b, the chloride channel calcium-activated 3 (Clca3), macrophage inflammatory protein (MIP) 1 alpha and 1 beta, eotaxin-2, serum amyloid A3 (Saa3), and insulin-like growth factor 1 (Igf1). These genes were induced to a greater extent in OVA-treated WT mice relative to ROR alpha(sg/sg) mice.Conclusions: Our study demonstrates that mice deficient in ROR alpha exhibit an attenuated allergic inflammatory response, indicating that ROR alpha plays a critical role in the development of Th2-driven allergic lung inflammation in mice, and suggests that this nuclear receptor should be further evaluated as a potential asthma target.

The retinoid-related orphan receptor alpha (RORalpha), a member of the ROR subfamily of nuclear receptors, has been implicated in the control of a number of physiological processes, including the regulation of several immune functions. To study the potential role of RORalpha in the regulation of innate immune responses in vivo, we analyzed the induction of airway inflammation in response to lipopolysaccharide (LPS) challenge in wild-type and staggerer (RORalpha(sg/sg)) mice, a natural mutant strain lacking RORalpha expression. Examination of hematoxylin and eosin-stained lung sections showed that RORalpha(sg/sg) mice displayed a higher degree of LPS-induced inflammation than wild-type mice. Bronchoalveolar lavage (BAL) was performed at 3, 16, and 24 h after LPS exposure to monitor the increase in inflammatory cells and the level of several cytokines/chemokines. The increased susceptibility of RORalpha(sg/sg) mice to LPS-induced airway inflammation correlated with a higher number of total cells and neutrophils in BAL fluids from LPS-treated RORalpha(sg/sg) mice compared with those from LPS-treated wild-type mice. In addition, IL-1beta, IL-6, and macrophage inflammatory protein-2 were appreciably more elevated in BAL fluids from LPS-treated RORalpha(sg/sg) mice compared with those from LPS-treated wild-type mice. The enhanced susceptibility of RORalpha(sg/sg) mice appeared not to be due to a repression of IkappaBalpha expression. Our observations indicate that RORalpha(sg/sg) mice are more susceptible to LPS-induced airway inflammation and are in agreement with the hypothesis that RORalpha functions as a negative regulator of LPS-induced inflammatory responses.

The retinoid-related orphan receptor α (RORα), a member of the ROR subfamily of nuclear receptors, has been implicated in the control of a number of physiological processes, including the regulation of several immune functions. To study the potential role of RORα in the regulation of innate immune responses in vivo, we analyzed the induction of airway inflammation in response to lipopolysaccharide (LPS) challenge in wild-type and staggerer (RORαsg/sg) mice, a natural mutant strain lacking RORα expression. Examination of hematoxylin and eosin-stained lung sections showed that RORαsg/sgmice displayed a higher degree of LPS-induced inflammation than wild-type mice. Bronchoalveolar lavage (BAL) was performed at 3, 16, and 24 h after LPS exposure to monitor the increase in inflammatory cells and the level of several cytokines/chemokines. The increased susceptibility of RORαsg/sgmice to LPS-induced airway inflammation correlated with a higher number of total cells and neutrophils in BAL fluids from LPS-treated RORαsg/sgmice compared with those from LPS-treated wild-type mice. In addition, IL-1β, IL-6, and macrophage inflammatory protein-2 were appreciably more elevated in BAL fluids from LPS-treated RORαsg/sgmice compared with those from LPS-treated wild-type mice. The enhanced susceptibility of RORαsg/sgmice appeared not to be due to a repression of IκBα expression. Our observations indicate that RORαsg/sgmice are more susceptible to LPS-induced airway inflammation and are in agreement with the hypothesis that RORα functions as a negative regulator of LPS-induced inflammatory responses.