SerpinB2 deficiency is associated with delayed mammary tumor development and decreased pro-tumorigenic macrophage polarization.

The in vivo functions of SerpinB2 in tumor cells and tumor-associated macrophages (TAMs) during breast cancer development and metastasis remain elusive. SerpinB2-deficient MMTV-PyMT mice (PyMTSB2-/-) were previously produced to explore the biological roles of SerpinB2 in breast cancer. Compared with MMTV-PyMT wild-type (PyMTWT) mice, PyMTSB2-/- mice showed delayed tumor progression and reduced CK8 + tumor cell dissemination to lymph nodes. RNA-Seq data revealed significantly enriched genes associated with inflammatory responses, especially upregulated M1 and downregulated M2 macrophage marker genes in PyMTSB2-/- tumors. Decreased CD206+M2 and increased NOS2+M1 markers were detected in the primary tumors and metastatic lymph nodes of PyMTSB2-/- mice. In an in vitro study, SerpinB2 knockdown decreased the sphere formation and migration of MDA-MB-231 cells and suppressed protumorigenic M2 polarization of RAW264.7 cells. The combination of low SerpinB2, high NOS2, and low CD206 expression was favorable for survival in patients with breast cancer, as assessed in the BreastMark dataset. Our study demonstrates that SerpinB2 deficiency delays mammary tumor development and metastasis in PyMTWT mice, along with reduced sphere formation and migration abilities of tumor cells and decreased macrophage protumorigenic polarization.

Estetrol/GPER/SERPINB2 transduction signaling inhibits the motility of triple-negative breast cancer cells.

BACKGROUND: Estetrol (E4) is a natural estrogen produced by the fetal liver during pregnancy. Due to its favorable safety profile, E4 was recently approved as estrogenic component of a new combined oral contraceptive. E4 is a selective ligand of estrogen receptor (ER)α and ERß, but its binding to the G Protein-Coupled Estrogen Receptor (GPER) has not been described to date. Therefore, we aimed to explore E4 action in GPER-positive Triple-Negative Breast Cancer (TNBC) cells. METHODS: The potential interaction between E4 and GPER was investigated by molecular modeling and binding assays. The whole transcriptomic modulation triggered by E4 in TNBC cells via GPER was explored through high-throughput RNA sequencing analyses. Gene and protein expression evaluations as well as migration and invasion assays allowed us to explore the involvement of the GPER-mediated induction of the plasminogen activator inhibitor type 2 (SERPINB2) in the biological responses triggered by E4 in TNBC cells. Furthermore, bioinformatics analysis was aimed at recognizing the biological significance of SERPINB2 in ER-negative breast cancer patients. RESULTS: After the molecular characterization of the E4 binding capacity to GPER, RNA-seq analysis revealed that the plasminogen activator inhibitor type 2 (SERPINB2) is one of the most up-regulated genes by E4 in a GPER-dependent manner. Worthy, we demonstrated that the GPER-mediated increase of SERPINB2 is engaged in the anti-migratory and anti-invasive effects elicited by E4 in TNBC cells. In accordance with these findings, a correlation between SERPINB2 levels and a good clinical outcome was found in ER-negative breast cancer patients. CONCLUSIONS: Overall, our results provide new insights into the mechanisms through which E4 can halt migratory and invasive features of TNBC cells.

Increased SERPINB2 potentiates 15LO1 expression via STAT6 signalling in epithelial cells in eosinophilic chronic rhinosinusitis with nasal polyps.

BACKGROUND: SERPINB2, a biomarker of Type-2 (T2) inflammatory processes, has been described in the context of asthma. Chronic rhinosinusitis with nasal polyps (CRSwNP) is also correlated with T2 inflammation and elevated 15LO1 induced by IL-4/13 in nasal epithelial cells. The aim of this study was to evaluate the expression and location of SERPINB2 in nasal epithelial cells (NECs) and determine whether SERPINB2 regulates 15LO1 and downstream T2 markers in NECs via STAT6 signalling. METHODS: SERPINB2 gene expression in bulk and single-cell RNAseq database was analysed by bioinformatics analysis. SERPINB2, 15LO1 and other T2 markers were evaluated from CRSwNP and HCs NECs. The colocalization of SERPINB2 and 15LO1 was evaluated by immunofluorescence. Fresh NECs were cultured at an air-liquid interface with or without IL-13, SERPINB2 Dicer-substrate short interfering RNAs (DsiRNAs) transfection, exogenous SERPINB2, 15-HETE recombinant protein and pSTAT6 inhibitors. 15LO1, 15-HETE and downstream T2 markers were analysed by qRT-PCR, western blot and ELISA. RESULTS: SERPINB2 expression was increased in eosinophilic nasal polyps compared with that in noneosinophilic nasal polyps and control tissues and positively correlated with 15LO1 and other downstream T2 markers. SERPINB2 was predominantly expressed by epithelial cells in NP tissue and was colocalized with 15LO1. In primary NECs in vitro, SERPINB2 expression was induced by IL-13. Knockdown or overexpression SERPINB2 decreased or enhanced expression of 15LO1 and 15-HETE in NECs, respectively, in a STAT6-dependent manner. SERPINB2 siRNA also inhibited the expression of the 15LO1 downstream genes, such as CCL26, POSTN and NOS2. STAT6 inhibition similarly decreased SERPINB2-induced 15LO1. CONCLUSIONS: SERPINB2 is increased in NP epithelial cells of eosinophilic CRSwNP (eCRSwNP) and contributes to T2 inflammation via STAT6 signalling. SERPINB2 could be considered a novel therapeutic target for eCRSwNP.

A microscale 3D organ on a chip for recapitulating reciprocal neuroendocrine crosstalk between the hypothalamus and the pituitary gland.

Conventional 2D or even recently developed 3Din vitroculture models for hypothalamus and pituitary gland cannot successfully recapitulate reciprocal neuroendocrine communications between these two pivotal neuroendocrine tissues known to play an essential role in controlling the body's endocrine system, survival, and reproduction. In addition, most currentvitroculture models for neuroendocrine tissues fail to properly reflect their complex multicellular structure. In this context, we developed a novel microscale chip platform, termed the 'hypothalamic-pituitary (HP) axis-on-a-chip,' which integrates various cellular components of the hypothalamus and pituitary gland with biomaterials such as collagen and hyaluronic acid. We used non-toxic blood coagulation factors (fibrinogen and thrombin) as natural cross-linking agents to increase the mechanical strength of biomaterials without showing residual toxicity to overcome drawbacks of conventional chemical cross-linking agents. Furthermore, we identified and verified SERPINB2 as a reliable neuroendocrine toxic marker, with its expression significantly increased in both hypothalamus and pituitary gland cells following exposure to various types of toxins. Next, we introduced SERPINB2-fluorescence reporter system into loaded hypothalamic cells and pituitary gland cells within each chamber of the HP axis on a chip, respectively. By incorporating this SERPINB2 detection system into the loaded hypothalamic and pituitary gland cells within our chip platform, Our HP axis-on-chip platform can better mimic reciprocal neuroendocrine crosstalk between the hypothalamus and the pituitary gland in the brain microenvironments with improved efficiency in evaluating neuroendocrine toxicities of certain drug candidates.

Unveiling the potential effects of acetylsalicylic acid: insights into regeneration in endometrial stem cells.

BACKGROUND: Although acetylsalicylic acid has been widely used for decades to treat and prevent various diseases, its potential effects on endometrial receptivity and subsequent pregnancy rates are still controversial due to conflicting data: many reports have shown positive effects of acetylsalicylic acid, whereas others have found that it has no effect. Furthermore, the direct effects of acetylsalicylic acid on various functions of normal endometrial cells, especially endometrial stem cells, and their underlying molecular mechanisms have not yet been proven. Recently, studies have revealed that a reduced number of active stem/progenitor cells within endometrial tissue limits cyclic endometrial regeneration and subsequently decreases pregnancy success rates, suggesting that endometrial stem cells play a critical role in endometrial regeneration and subsequent endometrial receptivity. METHODS: We assessed whether aspirin treatment can inhibit various endometrial stem cell functions related to regenerative capacity, such as self-renewal, migration, pluripotency/stemness, and differentiation capacity, in vitro. Next, we evaluated whether SERPINB2 regulates the effects of aspirin on endometrial stem cell functions by depleting SERPINB2 expression with specific shRNA targeting SERPINB2. To further investigate whether aspirin also inhibits various endometrial stem cell functions in vivo, aspirin was administered daily to mice through intraperitoneal (i.p.) injection for 7 days. RESULTS: In addition to its previously identified roles, to the best of our knowledge, we found for the first time that acetylsalicylic acid directly inhibits various human endometrial stem cell functions related to regenerative capacity (i.e., self-renewal, migration, differentiation, and capacity) through its novel target gene SERPINB2 in vitro. Acetylsalicylic acid exerts its function by suppressing well-known prosurvival pathways, such as Akt and/or ERK1/2 signaling, through a SERPINB2 signaling cascade. Moreover, we also found that acetylsalicylic acid markedly inhibits regenerative capacity-related functions in endometrial stem cells within tissue. CONCLUSIONS: We have found that acetylsalicylic acid has diverse effects on various endometrial stem cell functions related to regenerative capacity. Our findings are a critical step toward the development of more effective therapeutic strategies to increase the chances of successful pregnancy. Video Abstract.

Epithelial CST1 Promotes Airway Eosinophilic Inflammation in Asthma via the AKT Signaling Pathway.

PURPOSE: Epithelial cystatin SN (CST1), a type 2 cysteine protease inhibitor, was significantly upregulated in asthma. In this study, we aimed to investigate the potential role and mechanism of CST1 in eosinophilic inflammation in asthma. METHODS: Bioinformatics analysis on Gene Expression Omnibus datasets were used to explore the expression of CST1 in asthma. Sputum samples were collected from 76 asthmatics and 22 control subjects. CST1 mRNA and protein expression in the induced sputum were measured by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting. The possible function of CST1 was explored in ovalbumin (OVA)-induced eosinophilic asthma. Transcriptome sequencing (RNA-seq) was used to predict the possible regulated mechanism of CST1 in bronchial epithelial cells. Overexpression or knockdown of CST1 was further used to verify potential mechanisms in bronchial epithelial cells. RESULTS: CST1 expression was significantly increased in the epithelial cells and induced sputum of asthma. Increased CST1 was significantly associated with eosinophilic indicators and T helper cytokines. CST1 aggravated airway eosinophilic inflammation in the OVA-induced asthma model. In addition, overexpression of CST1 significantly enhanced the phosphorylation of AKT and the expression of serpin peptidase inhibitor, clade B, member 2 (SERPINB2), while knockdown using anti-CST1 siRNA reversed the trend. Furthermore, AKT had a positive effect on SERPINB2 expression. CONCLUSIONS: Increased sputum CST1 may play a key role in the pathogenesis of asthma through involvement in eosinophilic and type 2 inflammation through activation of the AKT signaling pathway, further promoting SERPINB2 expression. Therefore, targeting CST1 might be of therapeutic value in treating asthma with severe and eosinophilic phenotypes.

Therapeutic potential of nanoceria pretreatment in preventing the development of urological chronic pelvic pain syndrome: Immunomodulation via reactive oxygen species scavenging and SerpinB2 downregulation.

Urological chronic pelvic pain syndrome (UCPPS) manifests as pelvic pain with frequent urination and has a 10% prevalence rate without effective therapy. Nanoceria (cerium oxide nanoparticles [CNPs]) were synthesized in this study to achieve potential long-term pain relief, using a commonly used UCPPS mouse model with cyclophosphamide-induced cystitis. Transcriptome sequencing analysis revealed that serpin family B member 2 (SerpinB2) was the most upregulated marker in mouse bladder, and SerpinB2 was downregulated with CNP pretreatment. The transcriptome sequencing analysis results agreed with quantitative polymerase chain reaction and western blot analysis results for the expression of related mRNAs and proteins. Analysis of Gene Expression Omnibus (GEO) datasets revealed that SerpinB2 was a differentially upregulated gene in human UCPPS. In vitro SerpinB2 knockdown downregulated proinflammatory chemokine expression (chemokine receptor CXCR3 and C-X-C motif chemokine ligand 10) upon treatment with 4-hydroperoxycyclophosphamide. In conclusion, CNP pretreatment may prevent the development of UCPPS, and reactive oxygen species (ROS) scavenging and SerpinB2 downregulation may modulate the immune response in UCPPS.

SERPINB2, an Early Responsive Gene to Epigallocatechin Gallate, Inhibits Migration and Promotes Apoptosis in Esophageal Cancer Cells.

Esophageal cancer is a lethal disease that frequently occurs in developing countries, the incidence of which could be declined by drinking EGCG-enriched drinks or food. SERPINB2, whose complex functions and regulations are not yet fully understood, are induced by multiple inflammatory molecules and anti-tumor agents. Here, we identify 2444 EGCG-regulated genes in esophageal cancer cells, including SERPINB2. EGCG treatment recruits NF-κB at the promoter and enhancers of SERPINB2 and activates gene transcription, which is repressed by NF-κB knockdown or inhibition. Loss of SERPINB2 leads to a faster migration rate and less expression of Caspase-3 in cancer cells. Our study demonstrates that SERPINB2 is a new tumor-suppressor gene involved in cell movement and apoptosis and could be a therapeutic target for esophageal cancer.

The role of plasminogen activator inhibitor-2 in pneumococcal meningitis.

Pneumococcal meningitis is associated with dysregulation of the coagulation cascade. Previously, we detected upregulation of cerebral plasminogen activator inhibitor-2 (PAI-2) mRNA expression during pneumococcal meningitis. Diverse functions have been ascribed to PAI-2, but its role remains unclear. We analyzed the function of SERPINB2 (coding for PAI-2) in patients with bacterial meningitis, in a well-established pneumococcal meningitis mouse model, using Serpinb2 knockout mice, and in vitro in wt and PAI-2-deficient bone marrow-derived macrophages (BMDMs). We measured PAI-2 in cerebrospinal fluid of patients, and performed functional, histopathological, protein and mRNA expression analyses in vivo and in vitro. We found a substantial increase of PAI-2 concentration in CSF of patients with pneumococcal meningitis, and up-regulation and increased release of PAI-2 in mice. PAI-2 deficiency was associated with increased mortality in murine pneumococcal meningitis and cerebral hemorrhages. Serpinb2-/- mice exhibited increased C5a levels, but decreased IL-10 levels in the brain during pneumococcal infection. Our in vitro experiments confirmed increased expression and release of PAI-2 by wt BMDM and decreased IL-10 liberation by PAI-2-deficient BMDM upon pneumococcal challenge. Our data show that PAI-2 is elevated during in pneumococcal meningitis in humans and mice. PAI-2 deficiency causes an inflammatory imbalance, resulting in increased brain pathology and mortality.

miR-450b promotes cell migration and invasion by inhibiting SERPINB2 in oral squamous cell carcinoma.

OBJECTIVE: microRNA-450b (miR-450b) plays an important role in cancer progression; however, its function in oral squamous cell carcinoma (OSCC) remains largely unknown. This study aimed to investigate the action mechanisms of miR-450b in OSCC. MATERIALS AND METHODS: OSCC animal model was established via continuous induction with single-drug 7, 12-dimethylbenzo[a]anthracene (DMBA). Animal tissue samples were pathologically typed using haematoxylin-eosin (HE) staining. The Cancer Genome Atlas (TCGA) database was used to predict miR-450b and SERPINB2 expression in head and neck squamous cell carcinoma (HNSCC). qRT-PCR and Western blotting were used to detect gene and protein expression in OSCC tissue and cells, respectively. OSCC cell proliferation, growth, migration and invasion were detected using CCK-8, colony formation, transwell migration and matrigel invasion assays, respectively. Bioinformatic tools were used to predict miR-450b target genes. Dual-luciferase reporter assay was used to verify targeting between miR-450b and SERPINB2. Finally, small interfering RNA (siRNA) was used to reduce SERPINB2 expression to detect its effect on tumourigenesis. RESULTS: Four stages of OSCC carcinogenesis (normal oral epithelium, simple epithelial hyperplasia, dysplasia and OSCC) were identified. miR-450b was found to be overexpressed in OSCC animal samples, HNSCC samples and human OSCC cells. Upregulation of miR-450b significantly promoted OSCC cell proliferation, colony formation, migration and invasion, while its downregulation had the opposite effect. SERPINB2 was found to be a miR-450b target gene, and its expression was negatively correlated with miR-450b expression. Altering SERPINB2 expression effectively inhibited OSCC cell invasion, metastasis and epithelial-mesenchymal transition (EMT). CONCLUSIONS: miR-450b plays a key role in OSCC tumourigenesis by regulating OSCC cell migration, invasion and EMT via SERPINB2.

Effects of smoking on the tissue regeneration-associated functions of human endometrial stem cells via a novel target gene SERPINB2.

BACKGROUND: Smokers directly inhale mainstream cigarette smoke, which contains numerous known and potential toxic substances, and thus, smoking is expected to have broad harmful effects that cause tissue injury and dysfunction. Interestingly, many studies have suggested that the recent decline in female fertility and increased rate of spontaneous abortion could be associated with increased smoking rates. Indeed, women that smoked for 10 years or more were reported to have a ~ 20% higher infertility rate than women that had never smoked. However, the reasons for the underlying harmful aspects of smoking on female fertility remain a matter of debate. Importantly, a previous study revealed that resident endometrial stem cell deficiency significantly limits the cyclic regeneration potential of endometrium, which, in turn, decreases successful pregnancy outcomes. In this context, we postulated that exposure to mainstream cigarette smoke extracts might decrease female fertility by inhibiting the functions of resident endometrial stem cells. METHODS: We investigated whether cigarette mainstream smoke exposure directly inhibits various tissue regeneration-associated functions of endometrial stem cells, such as self-renewal, migration, pluripotency, and differentiation capacity in vitro. Next, we determined whether SERPINB2 mediates cigarette smoke-induced suppressive effects on various tissue regeneration-associated functions by depleting SERPINB2 expression with specific shRNA targeting SERPINB2. Mice were injected intraperitoneally with low (0.5 mg/kg) or high (1 mg/kg) doses of cigarette smoke extract (10 times for two weeks), and endometrial stem cells were then isolated from mice uterine tissues. RESULTS: We found that exposure to cigarette smoke extracts remarkably suppressed various tissue regeneration-associated functions of endometrial stem cells, such as self-renewal, migration, multilineage differentiation ability, and pluripotency in vitro and in vivo by activating the SERPINB2 gene. Indeed, cigarette smoke-induced inhibitory effects on various endometrial stem cell functions were significantly abolished by SERPINB2 knockdown. CONCLUSIONS: These findings provide valuable information on the harmful effects of cigarette smoking on resident endometrial stem cells and hopefully will facilitate the developments of promising therapeutic strategies for subfertile or infertile women that smoke cigarettes.

Transcriptome analysis of SerpinB2-deficient breast tumors provides insight into deciphering SerpinB2-mediated roles in breast cancer progression.

BACKGROUND: SerpinB2 is highly expressed in immune and tumor cells and is involved in multiple biological functions, including cell survival and remodeling for disease progression. This study prepared SerpinB2-deficient mice and analyzed the differentially expressed genes (DEGs) to determine if loss of this protein delays mammary tumor progression. RESULTS: A total of 305 DEGs (75 upregulated and 230 downregulated; > 1.5-fold difference, P < 0.05) were identified in SB2-/-;PyMT tumors compared with PyMT tumors. The DEGs were mainly involved in immune and inflammatory responses related to T cell differentiation, IFN-γ production, and lymphocyte chemotaxis based on 61 enriched GO terms, hierarchical clustering, KEGG pathways, and a functionally grouped annotation network. The significantly changed DEGs (Anxa3, Ccl17, Cxcl13, Cxcr3, IFN-γ, Nr4a1, and Sema3a) annotated with at least two GO categories in SB2-/-;PyMT tumors was validated by qRT-PCR. CONCLUSIONS: SerpinB2 deficiency alters the expression of multiple genes in mammary tumors, which might cause a delay in PyMT-induced mammary tumor progression.

A Novel Role for Plasminogen Activator Inhibitor Type-2 as a Hypochlorite-Resistant Serine Protease Inhibitor and Holdase Chaperone.

Plasminogen activator inhibitor type-2 (PAI-2), a member of the serpin family, is dramatically upregulated during pregnancy and in response to inflammation. Although PAI-2 exists in glycosylated and non-glycosylated forms in vivo, the majority of in vitro studies of PAI-2 have exclusively involved the intracellular non-glycosylated form. This study shows that exposure to inflammation-associated hypochlorite induces the oligomerisation of PAI-2 via a mechanism involving dityrosine formation. Compared to plasminogen activator inhibitor type-1 (PAI-1), both forms of PAI-2 are more resistant to hypochlorite-induced inactivation of its protease inhibitory activity. Holdase-type extracellular chaperone activity plays a putative non-canonical role for PAI-2. Our data demonstrate that glycosylated PAI-2 more efficiently inhibits the aggregation of Alzheimer's disease and preeclampsia-associated amyloid beta peptide (Aß), compared to non-glycosylated PAI-2 in vitro. However, hypochlorite-induced modification of non-glycosylated PAI-2 dramatically enhances its holdase activity by promoting the formation of very high-molecular-mass chaperone-active PAI-2 oligomers. Both PAI-2 forms protect against Aß-induced cytotoxicity in the SH-SY5Y neuroblastoma cell line in vitro. In the villous placenta, PAI-2 is localised primarily to syncytiotrophoblast with wide interpersonal variation in women with preeclampsia and in gestational-age-matched controls. Although intracellular PAI-2 and Aß staining localised to different placental cell types, some PAI-2 co-localised with Aß in the extracellular plaque-like aggregated deposits abundant in preeclamptic placenta. Thus, PAI-2 potentially contributes to controlling aberrant fibrinolysis and the accumulation of misfolded proteins in states characterised by oxidative and proteostasis stress, such as in Alzheimer's disease and preeclampsia.

Evi1 involved in benzene-induced haematotoxicity via modulation of PI3K/mTOR pathway and negative regulation Serpinb2.

Benzene is a widely used chemical and an environmental pollutant. Exposure to benzene can cause blood diseases, but the mechanisms underlying benzene haematotoxicity have not been fully clarified. Ecotropic virus integration site-1 (Evi1), a transcription factor, plays important roles in normal haematopoiesis and haematological diseases. In this study, we investigated the role and mechanism of Evi1 in benzene-induced haematotoxicity. We found that benzene exposure significantly increased Evi1 level in white blood cells (WBCs) in occupational benzene workers as well as mouse bone marrow cells. Further in vitro results demonstrated that compared with control cells exposed to same 1,4-benzoquinone (1,4-BQ, an important active metabolite of benzene) concentration, Evi1 downregulation significantly reduced cell proliferation, and disrupted cell viability, apoptosis, erythroid and megakaryotic cell differentiation and cell cycle. Additionally, down-regulation of Evi1 suppressed phosphoinositide 3-kinase (PI3K)/mTOR signalling pathway and elevated its target gene Serpinb2 following 1,4-BQ exposure. Moreover, the PI3K activator could partially relieve the inhibitory effect of down-regulation of Evi1 on cell proliferation and increase cell arrest in in G2/M phase. What's more, downregulation of Serpinb2 could partially alleviate proliferation inhibition and reverse cell cycle changes in G0/G1 phase and S phase induced by Evi1 inhibition. In conclusion, our data revealed that Evi1 downregulation aggravated the inhibition of cell proliferation and arrested cells in the G0/G1 phase when exposed to 1,4-BQ, potentially by inactivating the PI3K/mTOR pathway and upregulating downstream target gene Serpinb2. Our study provides novel insights on mechanism by which Evi1 participates in benzene-induced haematotoxicity.

Knockdown of SERPINB2 enhances the osteogenic differentiation of human bone marrow mesenchymal stem cells via activation of the Wnt/ß-catenin signalling pathway.

BACKGROUND: Globally, bone fractures are the most common musculoskeletal trauma, and approximately 8-10% of cases that fall into the categories of delayed or non-union healing. To date, there are no efficient pharmacological agents to accelerate the healing of bone fractures. Thus, it is necessary to find new strategies that accelerate bone healing and reduce the incidence of non-union or delayed fracture healing. Previous studies have revealed that the plasminogen activation system has been demonstrated to play an important role in bone metabolism. However, the function of SERPINB2 in the osteogenesis of hBMSCs remains unclear. Therefore, in this study, we investigated the effects and mechanism of SERPINB2 on osteogenic differentiation. METHODS: We investigated the osteogenesis effects of hBMSCs by both exogenous SerpinB2 protein and SERPINB2 gene silencing in vitro. Cell proliferation assay was used to assess the effect of exogenous SerpinB2 or SERPINB2 silencing on proliferation of hBMSCs. qPCR and Western blotting analysis detected the expression of target genes and proteins respectively. ALP staining was used to evaluated ALP activity and Alizarin Red staining (ARS) was used to evaluate mineral deposition. In vivo, a murie tibial fracture model was established, histological evaluation and radiographic analysis was used to confirm the therapeutic effects of SERPINB2 silencing in fracture healing. Statistical significance between two groups was determined by Student's t test, one-way ANOVA or Bonferroni's post-hoc test according to the distribution of the tested population. RESULTS: The addition of exogenous SerpinB2 protein inhibted osteoblast differentiation of hBMSCs in vitro, while SERPINB2 gene silencing significant promote osteoblast differentiation of hBMSCs in vitro. And silenced SERPINB2 gene also increased mineral deposits. Moreover, ß-catenin levels were up-regulated by SERPINB2 gene depletion. And the enhancement of osteogenic differentiation induced by SERPINB2 silencing was almost inhibited by specific Wnt/ß-catenin signaling pathway inhibitor. In a murine tibial fracture model, local injection of SERPINB2 siRNA improved bone fracture healing. CONCLUSIONS: Taken together, these findings indicate that SERPINB2 silencing promoted osteogenic differentiation of BMSCs via the Wnt/ß-catenin signaling pathway, and silenced SERPINB2 in vivo effectively promotes fracture healing, suggesting that SERPINB2 may be a novel target for bone fracture healing.

SERPINB2-its regulation and interplay with aryl hydrocarbon receptor.

Despite many years of intensive investigation, real biological role of SERPINB2 is largely unknown. However, recent high throughput studies suggest its function in inflammation, influence on autoimmune disorders, and modulation of processes leading to carcinogenesis. SERPINB2 expression is acutely upregulated by many different stimuli, among others by aryl hydrocarbon receptor ligands. Mechanisms of regulation of SERPINB2 expression, involvement of the gene in processes leading to inflammation or carcinogenesis, and its interplay with aryl hydrocarbon receptor are subject of present review.

SERPINA9 and SERPINB2: Novel Cartilage Lineage Differentiation Markers of Human Mesenchymal Stem Cells with Kartogenin.

OBJECTIVE: Human mesenchymal stem cells (hMSCs) are a promising source for regenerative medicine, especially mesodermal lineages. Clinical applications require an understanding of the mechanisms for transcriptional control to maintain the desired cell type. The aim of this study was to identify novel markers for differentiation of hMSCs into bone or cartilage with the use of Kartogenin, by RNA analysis using microarray technology, and explore the role of RhoA-Rho associated protein kinase (ROCK) inhibition in these. METHODS: Commercial human bone marrow derived primary mesenchymal stem cells were purchased from ATCC. Cells were differentiated in vitro in 2-dimensional cultures using Kartogenin as the main cartilage inducer and bone morphogenetic protein 2 for bone differentiation; cells were cultured with and without ROCK inhibitor Y-27632. After 21 days of culture, whole RNA was extracted and analyzed via Affimetrix microarrays. The most significant hits were validated by quantitative polymerase chain reaction. RESULTS: We found a total of 1,757 genes that were either up- or downregulated on differentiation, when compared to P1 hMSC (control) at day 0 of differentiation. Two members of the Serpin superfamily, SERPINA9 and SERPINB2, were significantly upregulated in the cartilage groups, whereas they were unchanged in the bone groups with and without ROCK inhibition. CONCLUSIONS: SERPINA9 and SERPINB2 are novel differentiation markers, and molecular regulator candidates for hMSC lineage commitment toward bone and cartilage, providing a new tool for regenerative medicine. Our study highlights the roles of these 2 genes, with significant upregulation of both in cell cultures stimulated with Kartogenin.

N-Alkylisatin-Loaded Liposomes Target the Urokinase Plasminogen Activator System in Breast Cancer.

The urokinase plasminogen activator and its receptor (uPA/uPAR) are biomarkers for metastasis, especially in triple-negative breast cancer. We prepared anti-mitotic N-alkylisatin (N-AI)-loaded liposomes functionalized with the uPA/uPAR targeting ligand, plasminogen activator inhibitor type 2 (PAI-2/SerpinB2), and assessed liposome uptake in vitro and in vivo. Receptor-dependent uptake of PAI-2-functionalized liposomes was significantly higher in the uPA/uPAR overexpressing MDA-MB-231 breast cancer cell line relative to the low uPAR/uPAR expressing MCF-7 breast cancer cell line. Furthermore, N-AI cytotoxicity was enhanced in a receptor-dependent manner. In vivo, PAI-2 N-AI liposomes had a plasma half-life of 5.82 h and showed an increased accumulation at the primary tumor site in an orthotopic MDA-MB-231 BALB/c-Fox1nu/Ausb xenograft mouse model, relative to the non-functionalized liposomes, up to 6 h post-injection. These findings support the further development of N-AI-loaded PAI-2-functionalized liposomes for uPA/uPAR-positive breast cancer, especially against triple-negative breast cancer, for which the prognosis is poor and treatment is limited.

TNFα-Erk1/2 signaling pathway-regulated SerpinE1 and SerpinB2 are involved in lipopolysaccharide-induced porcine granulosa cell proliferation.

Lipopolysaccharide (LPS) is an inhibitory factor that causes hormonal imbalance and subsequently affects ovarian function and fertility in mammals. Previous studies have shown that the exposure of granulosa cells (GC) to LPS leads to steroidogenesis dysfunction. However, the effects of LPS on the viability of GC remain largely unclear. In the present study, we aimed to address this question and unveil the underlying molecular mechanisms using cultured porcine GC. Results showed that GC proliferation and tumor necrosis factor α (TNFα) secretion were significantly increased after exposure to LPS, and these effects were completely reversed by blocking the TNFα sheddase, ADAM17. Moreover, GC proliferation induced by LPS was mimicked by treatment with recombinant TNFα. In addition, SerpinE1 and SerpinB2 expression levels increased in GC after treatment with LPS or recombinant TNFα, whereas blocking the Erk1/2 pathway completely abolished these effects and also inhibited GC proliferation. Further, consistent with the effects of blocking the Erk1/2 pathway, cell proliferation was completely inhibited by knocking down SerpinE1 or SerpinB2 in the presence of LPS or recombinant TNFα. Mitochondrial membrane potential (MMP) polarization in GC was increased by LPS or recombinant TNFα treatment, and these changes were completely negated by Erk1/2 inhibition, but not by SerpinE1 or SerpinB2 knockdown. Taken together, these results suggested that the TNFα-mediated upregulation of SerpinE1 and SerpinB2, through activation of the Erk1/2 pathway plays a crucial role in LPS-stimulated GC proliferation, and the increase in GC MMP may synergistically influence this process.

Expression of Serpin Peptidase Inhibitor B2 (SERPINB2) is regulated by Aryl hydrocarbon receptor (AhR).

Aryl hydrocarbon receptor (AhR) is a highly conserved ligand-activated transcription factor with high affinity to aromatic planar compounds, such as ß-naphthoflavone (BNF), benzo[a]pyrene (BaP) or dioxin (TCDD). After binding the ligand, AhR triggers induction of the expression of phase I and phase II drug-metabolizing genes, together with numerous other genes that are not directly involved in the metabolism of xenobiotics. Several studies have shown that AhR plays a role in tumor initiation, promotion and progression, but the molecular mechanisms involved in these processes are not fully understood. A previous study from our laboratory indicated that the SERPINB2 gene is presumably regulated by AhR. To prove that such induction is really AhR-dependent, in the present study we knocked down the expression of AhR by stable transfection of a laryngeal squamous cell carcinoma cell line (UT-SCC-34) with shRNA, resulting in 92% reduction of BNF-induced expression of SERPINB2. However, in silico analysis did not reveal AhR-dependent responsive elements in the promoter of the SERPINB2 gene. Therefore, to address this problem, we have used cycloheximide, an inhibitor of translation, and our results clearly indicate that an additional, newly synthesized protein is involved in AhR-dependent induction of SERPINB2 expression by BNF. So, to exclude that AhR binds to the putative xenobiotic-responsive elements (XREs) localized upstream of the SERPINB2 gene, we performed chromatin immunoprecipitation assays. As expected, we found no direct binding of AhR to its responsive elements in the vicinity of the SERPINB2 gene, further demonstrating the indirect SERPINB2 induction by AhR. However, the further analysis demonstrated that the expression of the enhancer RNA encoded by the region of DNA 20 kbp upstream from the SERPINB2 gene was AhR-dependent. Although AhR-mediated SERPINB2 induction clearly requires the synthesis of an additional protein, the kinetics of SERPINB2 induction is as fast as the kinetics of CYP1A1 and CYP1B1 induction (both genes directly regulated by AhR). Therefore, given previous studies regarding the induction of SERPINB2 expression by bacterial lipopolysaccharides (LPS), we think that, similarly, the interaction with pause-release proteins may be responsible for AhR-dependent regulation of SERPINB2 expression.