Secretoglobin 3A2 protects lung from developing cigarette smoke-induced pulmonary emphysema.

Secretoglobin (SCGB) 3A2 is a bioactive molecule exhibiting various functions such as improving allergic airway inflammation and pulmonary fibrosis and promoting bronchial branching and proliferation during lung development. To determine if and how SCGB3A2 is involved in chronic obstructive pulmonary disease (COPD), a multifactorial disease with both airway and emphysematous lesions, a COPD mouse model was created by exposing Scgb3a2-deficient (KO), Scgb3a2-lung-specific overexpressing (TG), and wild type (WT) mice to cigarette smoke (CS) for 6 months. The KO mice showed loss of lung structure under control condition, and CS exposure resulted in more expansion of airspace and destruction of alveolar wall than WT mouse lungs. In contrast, TG mouse lungs showed no significant changes after CS exposure. SCGB3A2 increased the expression and phosphorylation of signal transducers and activators of transcription (STAT)1 and STAT3, and the expression of α1-antitrypsin (A1AT) in mouse lung fibroblast-derived MLg cells and mouse lung epithelial-derived MLE-15 cells. In MLg cells, A1AT expression was decreased in Stat3-knockdown cells, and increased upon Stat3 overexpression. STAT3 formed a homodimer when cells were stimulated with SCGB3A2. Chromatin immunoprecipitation and reporter assays demonstrated that STAT3 binds to specific binding sites on the Serpina1a gene encoding A1AT and upregulates its transcription in lung tissues of mice. Furthermore, nuclear localization of phosphorylated STAT3 upon SCGB3A2 stimulation was detected by immunocytochemistry. These findings demonstrate that SCGB3A2 protects the lungs from the development of CS-induced emphysema by regulating A1AT expression through STAT3 signaling.

Development of Aging-Related Emphysematous and Lymphoma-Like Lesions is Enhanced by the Lack of Secretoglobin 3A2 in Mouse Lungs.

Background: Secretoglobin (SCGB) 3A2 is a novel bioactive molecule with anti-inflammatory and anti-fibrotic activities. SCGB3A2 also promotes the maturation of bronchial divergence and the lungs during embryonic development. However, much remains unknown concerning the roles of SCGB3A2 in diseases associated with aging. Methods: The lungs of Scgb3a2-knockout (KO) mice and their wild-type (WT) littermates were subjected to histological analysis, Victoria blue staining to evaluate of elastic fibers, and lung morphometric analysis during the postnatal period (birth to 8 weeks) and during aging (8 weeks to 2 years). Their spleens were also histologically evaluated. The expression of lung surfactant protein (SP) mRNAs was examined by quantitative reverse transcriptase-polymerase chain reaction. RNA sequencing (RNAseq) analysis was performed on 3-month-old KO and WT mouse lungs. Results: The alveolar spaces of KO mice continuously expanded between 0.5 and 2 years of age, accompanied by increases of the mean linear intercept and destructive index. KO mouse lungs displayed inflammation associated with lymphocyte aggregate starting at 1 year of age, and the inflammation was worse than that of WT mouse lungs. A high number of lymphoma-like cells were presented in 2-year-old KO mouse lungs. White pulp fusion was detected in the spleens of both WT and KO mice older than 0.5 years; however, the fusion was more severe in KO mice than in WT mice. The expression of surfactant protein (SP)-A, SP-B, SP-C, and SP-D mRNAs in KO mouse lungs decreased with age, and after 1 year of age, the expression of most SPs was significantly lower in KO mice than in WT mice. RNAseq demonstrated that the expression of immune system-related genes was highly altered in KO mouse lungs. Conclusion: SCGB3A2 may be required for maintaining homeostasis and immune activity in the lungs during aging. SCGB3A2 deficiency might increase the risk of emphysema of the lung.

Heterozygous SOD1 deficiency in mice with an NZW background causes male infertility and an aberrant immune phenotype.

New Zealand white (NZW) mouse is a mutant strain that has a larval defect in the immune system, and a F1 hybrid between NZW and New Zealand Black mouse spontaneously develops systemic lupus erythematosus (SLE). In meantime, the ablation of superoxide dismutase 1 (Sod1) causes autoimmune haemolytic anaemia, a clinical condition of SLE, in mice with a C57BL/6 background. On the basis of our previous studies, we hypothesised that oxidative stress may trigger this aberrant autoimmunity in NZW mice without crossing with another strain. To validate this, we attempted to establish Sod1-/-/NZW mice but this attempt failed to obtain any objective mouse. The congenic Sod1+/-/NZW male mice were completely infertile because of severe oligozoospermia attributed to a defect in spermatogenesis. The levels of the SOD1 protein were about a half in the testes of the Sod1+/-/NZW mice. Sperm from the Sod1+/-/NZW mice were largely defective and showed quite low fertilising ability in in vitro fertilisation assays. Concomitant with an increase in the oxidatively modified proteins, spermatogenic cells underwent more cell death in the testes of the Sod1+/-/NZW mice compared to those of WT/NZW mice. An examination of immunocompetent cells from Sod1+/-/NZW mice indicated an abnormality in T-cell responses. These collective results suggest that the oxidative stress caused by an SOD1 haploinsufficiency exerts deleterious effects on the testis, either directly on spermatogenic cells or via the destabilisation of the autoimmune response in Sod1+/-/NZW mice.

SCGB3A2 Inhibits Acrolein-Induced Apoptosis through Decreased p53 Phosphorylation.

Chronic obstructive pulmonary disease (COPD), a major global health problem with increasing morbidity and mortality rates, is anticipated to become the third leading cause of death worldwide by 2020. COPD arises from exposure to cigarette smoke. Acrolein, which is contained in cigarette smoke, is the most important risk factor for COPD. It causes lung injury through altering apoptosis and causes inflammation by augmenting p53 phosphorylation and producing reactive oxygen species (ROS). Secretoglobin (SCGB) 3A2, a secretory protein predominantly present in the epithelial cells of the lungs and trachea, is a cytokine-like small molecule having anti-inflammatory, antifibrotic, and growth factor activities. In this study, the effect of SCGB3A2 on acrolein-related apoptosis was investigated using the mouse fibroblast cell line MLg as the first step in determining the possible therapeutic value of SCGB3A2 in COPD. Acrolein increased the production of ROS and phosphorylation of p53 and induced apoptosis in MLg cells. While the extent of ROS production induced by acrolein was not affected by SCGB3A2, p53 phosphorylation was significantly decreased by SCGB3A2. These results demonstrate that SCGB3A2 inhibited acrolein-induced apoptosis through decreased p53 phosphorylation, not altered ROS levels.

Macrophage colony-stimulating factor induces prolactin expression in rat pituitary gland.

We investigated the role of macrophage colony-stimulating factor (M-CSF) in the pituitary gland to understand the effect of M-CSF on pituitary hormones and the relationship between the endocrine and immune systems. When we attempted to establish pituitary cell lines from a thyrotropic pituitary tumor (TtT), a macrophage cell line, TtT/M-87, was established. We evaluated M-CSF-like activity in conditioned media (CM) from seven pituitary cell lines using TtT/M-87 cells. TtT/M-87 proliferation significantly increased in the presence of CM from TtT/GF cells, a pituitary folliculostellate (FS) cell line. M-CSF mRNA was detected in TtT/GF and MtT/E cells by reverse transcriptase-polymerase chain reaction (RT-PCR), and its expression in TtT/GF cells was increased in a lipopolysaccharide (LPS) dose-dependent manner. M-CSF mRNA expression was also increased in rat anterior pituitary glands by LPS. M-CSF receptor (M-CSFR) mRNA was only detected in TtT/ M-87 cells and increased in the LPS-stimulated rat pituitary glands. In rat pituitary glands, M-CSF and M-CSFR were found to be localized in FS cells and prolactin (PRL)-secreting cells, respectively, by immunohistochemistry. The PRL concentration in rat sera was significantly increased at 24 h after M-CSF administration, and mRNA levels significantly increased in primary culture cells of rat anterior pituitary glands. In addition, TNF-α mRNA was increased in the primary culture cells by M-CSF. These results revealed that M-CSF was secreted from FS cells and M-CSF regulated PRL expression in rat pituitary glands.

Physiological characteristics of gastric contractions and circadian gastric motility in the free-moving conscious house musk shrew (Suncus murinus).

Although many studies have demonstrated the physiological action of motilin on the migrating motor complex, the precise mechanisms remain obscure. To obtain new insights into the mechanisms, we focused on the house musk shrew (Suncus murinus, suncus used as a laboratory name) as a small model animal for in vivo motilin study, and we studied the physiological characteristics of suncus gastrointestinal motility. Strain gauge transducers were implanted on the serosa of the gastric body and duodenum, and we recorded gastrointestinal contractions in the free-moving conscious suncus and also examined the effects of intravenous infusion of various agents on gastrointestinal motility. During the fasted state, the suncus stomach and duodenum showed clear migrating phase III contractions (intervals of 80-150 min) as found in humans and dogs. Motilin (bolus injection, 100-300 ng/kg; continuous infusion, 10-100 ng·kg(-1)·min(-1)) and erythromycin (80 µg·kg(-1)·min(-1)) induced gastric phase III contractions, and motilin injection also increased the gastric motility index in a dose-dependent manner (P < 0.05, vs. saline). Pretreatment with atropine completely abolished the motilin-induced gastric phase III contractions. On the other hand, in the free-feeding condition, the suncus showed a relatively long fasting period in the light phase followed by spontaneous gastric phase III contractions. The results suggest that the suncus has almost the same gastrointestinal motility and motilin response as those found in humans and dogs, and we propose the suncus as a new small model animal for studying gastrointestinal motility and motilin in vivo.

Identification of ghrelin in the house musk shrew (Suncus murinus): cDNA cloning, peptide purification and tissue distribution.

Ghrelin is the endogenous ligand for the growth hormone (GH) secretagogue receptor, and the sequence of ghrelin has been determined in many species from fish to mammals. In the present study, to reveal the production of ghrelin in the house musk shrew (Suncus murinus, order: Insectivora, suncus is used as a laboratory name), we determined the cDNA sequence and structure of suncus ghrelin and also demonstrated the ghrelin-producing cells in the gastrointestinal tract. Results of cDNA cloning and mass spectrometry analysis revealed that suncus ghrelin is composed of 18 or 26 amino acid residues and that the 3rd Ser was acylated mainly by n-octanoic acid. The 10 amino acids of the N-terminal region of suncus mature ghrelin were consistent with those of other mammals. Quantitative RT-PCR revealed that suncus ghrelin mRNA is highly expressed in the gastric corpus and pyloric antrum, and low expression levels were found in various tissues, including the intestinal tract. Ghrelin cells were found only in the corpus and antrum by immunohistochemistry and in situ hybridization, and most of the ghrelin cells were closed-type cells with relatively rich cytoplasm and scattered in the glandular body and base of the gastric mucosa. The density of ghrelin cells in the corpus was significantly greater than that in the antrum. The results of this study together with our recent results regarding motilin production in the suncus indicate that the suncus will be a useful model animal for study of physiological function of the motilin/ghrelin family.