Serpins promote cancer cell survival and vascular co-option in brain metastasis.

Brain metastasis is an ominous complication of cancer, yet most cancer cells that infiltrate the brain die of unknown causes. Here, we identify plasmin from the reactive brain stroma as a defense against metastatic invasion, and plasminogen activator (PA) inhibitory serpins in cancer cells as a shield against this defense. Plasmin suppresses brain metastasis in two ways: by converting membrane-bound astrocytic FasL into a paracrine death signal for cancer cells, and by inactivating the axon pathfinding molecule L1CAM, which metastatic cells express for spreading along brain capillaries and for metastatic outgrowth. Brain metastatic cells from lung cancer and breast cancer express high levels of anti-PA serpins, including neuroserpin and serpin B2, to prevent plasmin generation and its metastasis-suppressive effects. By protecting cancer cells from death signals and fostering vascular co-option, anti-PA serpins provide a unifying mechanism for the initiation of brain metastasis in lung and breast cancers.

Capsaicin enhances cisplatin-induced anti-metastasis of nasopharyngeal carcinoma by inhibiting EMT and ERK signaling via serpin family B member 2.

Cisplatin (DDP)-based combined chemotherapy or concurrent chemoradiotherapy is the mainstay treatment for advanced-stage nasopharyngeal carcinoma (NPC), but needs improvement due to its severe side effects. Capsaicin (CAP) can enhance the anti-tumor activity of cytotoxic drugs. The aim of this study was to investigate the anti-metastasis activity of CAP in combination with DDP in NPC. Herein, CAP and DDP showed synergistic cytotoxic effects on NPC cells. CAP alone and DDP alone inhibited NPC migration and invasion in vitro and in vivo, and the combination of CAP and DDP had the greatest effect. Moreover, CAP upregulated the mRNA and protein expressions of serpin family B member 2 (SERPINB2). Further results showed that both SERPINB2 mRNA and protein expressions were downregulated in NPC cell lines and tissues and SERPINB2 overexpression inhibited NPC migration and invasion in vitro and in vivo, while silencing SERPINB2 acted oppositely. In addition, SERPINB2 was abnormally expressed in head and neck squamous cell carcinoma and other multiple cancers, and downregulation of SERPINB2 predicted poor prognosis in head and neck squamous cell carcinoma according to the Cancer Genome Atlas database. We further found that SERPINB2 overexpression inhibited epithelial-mesenchymal transition (EMT) and the phosphorylated extracellular signal-regulated kinase (p-ERK), and the inhibitory effect was enhanced by CAP and DDP. Altogether, our results suggest that the combined inhibition of CAP and DDP on NPC metastasis may be related to the inhibition of epithelial-mesenchymal transition and ERK signals mediated by SERPINB2, and CAP may help to improve the efficacy of DDP in the treatment of NPC and develop new therapeutic approaches.

PAI-1 deficiency drives pulmonary vascular smooth muscle remodeling and pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by vasoconstriction and remodeling of small pulmonary arteries (PAs). Central to the remodeling process is a switch of pulmonary vascular cells to a proliferative, apoptosis-resistant phenotype. Plasminogen activator inhibitors-1 and -2 (PAI-1 and PAI-2) are the primary physiological inhibitors of urokinase-type and tissue-type plasminogen activators (uPA and tPA), but their roles in PAH are unsettled. Here, we report that: 1) PAI-1, but not PAI-2, is deficient in remodeled small PAs and in early-passage PA smooth muscle and endothelial cells (PASMCs and PAECs) from subjects with PAH compared with controls; 2) PAI-1-/- mice spontaneously develop pulmonary vascular remodeling associated with upregulation of mTORC1 signaling, pulmonary hypertension (PH), and right ventricle (RV) hypertrophy; and 3) pharmacological inhibition of uPA in human PAH PASMCs suppresses proproliferative mTORC1 and SMAD3 signaling, restores PAI-1 levels, reduces proliferation, and induces apoptosis in vitro, and prevents the development of SU5416/hypoxia-induced PH and RV hypertrophy in vivo in mice. These data strongly suggest that downregulation of PAI-1 in small PAs promotes vascular remodeling and PH due to unopposed activation of uPA and consequent upregulation of mTOR and transforming growth factor-ß (TGF-ß) signaling in PASMCs, and call for further studies to determine the potential benefits of targeting the PAI-1/uPA imbalance to attenuate and/or reverse pulmonary vascular remodeling and PH.NEW & NOTEWORTHY This study identifies a novel role for the deficiency of plasminogen activator inhibitor (PAI)-1 and resultant unrestricted uPA activity in PASMC remodeling and PH in vitro and in vivo, provides novel mechanistic link from PAI-1 loss through uPA-induced Akt/mTOR and TGFß-Smad3 upregulation to pulmonary vascular remodeling in PH, and suggests that inhibition of uPA to rebalance the uPA-PAI-1 tandem might provide a novel approach to complement current therapies used to mitigate this pulmonary vascular disease.

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.

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.

Role of different mechanisms in pro-inflammatory responses triggered by traffic-derived particulate matter in human bronchiolar epithelial cells.

BACKGROUND: Traffic-derived particles are important contributors to the adverse health effects of ambient particulate matter (PM). In Nordic countries, mineral particles from road pavement and diesel exhaust particles (DEP) are important constituents of traffic-derived PM. In the present study we compared the pro-inflammatory responses of mineral particles and DEP to PM from two road tunnels, and examined the mechanisms involved. METHODS: The pro-inflammatory potential of 100 µg/mL coarse (PM10-2.5), fine (PM2.5-0.18) and ultrafine PM (PM0.18) sampled in two road tunnels paved with different stone materials was assessed in human bronchial epithelial cells (HBEC3-KT), and compared to DEP and particles derived from the respective stone materials. Release of pro-inflammatory cytokines (CXCL8, IL-1α, IL-1ß) was measured by ELISA, while the expression of genes related to inflammation (COX2, CXCL8, IL-1α, IL-1ß, TNF-α), redox responses (HO-1) and metabolism (CYP1A1, CYP1B1, PAI-2) was determined by qPCR. The roles of the aryl hydrocarbon receptor (AhR) and reactive oxygen species (ROS) were examined by treatment with the AhR-inhibitor CH223191 and the anti-oxidant N-acetyl cysteine (NAC). RESULTS: Road tunnel PM caused time-dependent increases in expression of CXCL8, COX2, IL-1α, IL-1ß, TNF-α, COX2, PAI-2, CYP1A1, CYP1B1 and HO-1, with fine PM as more potent than coarse PM at early time-points. The stone particle samples and DEP induced lower cytokine release than all size-fractionated PM samples for one tunnel, and versus fine PM for the other tunnel. CH223191 partially reduced release and expression of IL-1α and CXCL8, and expression of COX2, for fine and coarse PM, depending on tunnel, response and time-point. Whereas expression of CYP1A1 was markedly reduced by CH223191, HO-1 expression was not affected. NAC reduced the release and expression of IL-1α and CXCL8, and COX2 expression, but augmented expression of CYP1A1 and HO-1. CONCLUSIONS: The results indicate that the pro-inflammatory responses of road tunnel PM in HBEC3-KT cells are not attributed to the mineral particles or DEP alone. The pro-inflammatory responses seem to involve AhR-dependent mechanisms, suggesting a role for organic constituents. ROS-mediated mechanisms were also involved, probably through AhR-independent pathways. DEP may be a contributor to the AhR-dependent responses, although other sources may be of importance.

Plasminogen activator Inhibitor-2 inhibits pulmonary arterial smooth muscle cell proliferation in pulmonary arterial hypertension via PI3K/Akt and ERK signaling.

BACKGROUND: The proliferation of pulmonary arterial smooth muscle cells (PASMCs) and subsequent pulmonary vascular remodeling leads to pulmonary arterial hypertension (PAH). Understanding the underlying mechanisms and identifying molecules that can suppress PASMCs proliferation is critical for developing effective pharmacological treatment. We previously showed that plasminogen activator inhibitor-2 (PAI-2) inhibited human PASMC (hPASMCs) proliferation in vitro. However, its inhibitory effect on PAH remains to be determined, and the mechanism remains to be illustrated. METHODS: We compared serum PAI-2 levels between PAH patients and healthy controls, and examined the correlation between PAI-2 level and disease severity. In monocrotaline-induced PAH rats, we examined the effects of exogenous PAI-2 administration on pulmonary vascular remodeling and PAH development. The effect of PAI-2 and potential mechanisms was further examined in cultured hPASMCs. RESULTS: The serum PAI-2 was decreased in PAH patients compared with controls. PAI-2 level was negatively correlated with mean pulmonary arterial pressure and estimated systolic pulmonary arterial pressure in ultrasonic cardiogram, while positively correlated with 6-min walking distance. In rats, administration of exogenous PAI-2 significantly reversed monocrotaline-induced PAH, as indicated by the decrease in right ventricle systolic pressure, right ventricular hypertrophy index and percent media thickness of pulmonary arterioles. Further mechanistic investigation in hPASMCs showed that PAI-2 inhibited cell proliferation by preventing the activation of PI3K/Akt and ERK pathways. CONCLUSION: PAI-2 is downregulated in PAH patients. PAI-2 attenuates PAH development by suppressing hPASMCs proliferation via the inhibition of PI3K/Akt and ERK pathways. PAI-2 may serve as a potential biomarker and therapeutic target for PAH.

Zinc Favors Triple-Negative Breast Cancer's Microenvironment Modulation and Cell Plasticity.

Triple-negative breast cancer (TNBC) tends to metastasize to the brain, a step that worsens the patient's prognosis. The specific hallmarks that determine successful metastasis are motility and invasion, microenvironment modulation, plasticity, and colonization. Zinc, an essential trace element, has been shown to be involved in all of these processes. In this work, we focus our attention on the potential role of zinc during TNBC metastasis. We used MDA-MB-BrM2 (BrM2) cells, a brain metastasis model derived from the parental TNBC cell line MDA-MB-231. Our studies show that BrM2 cells had double the zinc content of MDA-MB-231 cells. Moreover, exploring different metastatic hallmarks, we found that the zinc concentration is especially important in the microenvironment modulation of brain metastatic cells, enhancing the expression of SerpinB2. Furthermore, we show that zinc promotes the tumorigenic capacity of breast cancer stem cells. In addition, by causing a disturbance in MDA-MB-231 zinc homeostasis by overexpressing the Zip4 transporter, we were able to increase tumorigenicity. Nevertheless, this strategy did not completely recapitulate the BrM2 metastatic phenotype. Altogether, our work suggests that zinc plays an important role in the transformative steps that tumoral cells take to acquire tumorigenic potential and niche specificity.

Mycobacterium tuberculosis blocks crosslinking of annexin-1 and apoptotic envelope formation on infected macrophages to maintain virulence.

Macrophages infected with attenuated Mycobacterium tuberculosis strain H37Ra become apoptotic, which limits bacterial replication and facilitates antigen presentation. Here we demonstrate that cells infected with H37Ra became apoptotic after the formation of an apoptotic envelope on their surface was complete. This process required exposure of phosphatidylserine on the cell surface, followed by deposition of the phospholipid-binding protein annexin-1 and then transglutaminase-mediated crosslinking of annexin-1 through its amino-terminal domain. In macrophages infected with the virulent strain H37Rv, in contrast, the amino-terminal domain of annexin-1 was removed by proteolysis, thus preventing completion of the apoptotic envelope, which resulted in macrophage death by necrosis. Virulent M. tuberculosis therefore avoids the host defense system by blocking formation of the apoptotic envelope, which leads to macrophage necrosis and dissemination of infection in the lung.

An Endogenous Anti-aging Factor, Sonic Hedgehog, Suppresses Endometrial Stem Cell Aging through SERPINB2.

Endometrial stem cells are located in the basal layer of the endometrium, and they are responsible for the cyclic regeneration of the uterus during the menstrual cycle. Recent studies have revealed that recurrent pregnancy loss is associated with an age-related stem cell deficiency in the endometrium. Therefore, intensive study of endometrial stem cell aging may provide new insights for preventing recurrent pregnancy loss. Sonic hedgehog (SHH) signaling has been identified as a morphogen during the embryonic development processes. In addition to this canonical function, we found that the age-associated decline in regenerative potential in the endometrium may be due to decreased SHH-signaling integrity in local stem cells with aging. Importantly, the current study also showed that SHH activity clearly declines with aging both in vitro and in vivo, and exogenous SHH treatment significantly alleviates various aging-associated declines in multiple endometrial stem cell functions, suggesting that SHH may act as an endogenous anti-aging factor in human endometrial stem cells. Moreover, we found that stem cell senescence may enhance SERPINB2 expression, which in turn mediates the effect of SHH on alleviating senescence-induced endometrial stem cell dysfunctions, suggesting that SERPINB2 is a master regulator of SHH signaling during the aging process.

SASP-Dependent Interactions between Senescent Cells and Platelets Modulate Migration and Invasion of Cancer Cells.

Alterations in platelet aggregation are common in aging individuals and in the context of age-related pathologies such as cancer. So far, however, the effects of senescent cells on platelets have not been explored. In addition to serving as a barrier to tumor progression, cellular senescence can contribute to remodeling tissue microenvironments through the capacity of senescent cells to synthesize and secrete a plethora of bioactive factors, a feature referred to as the senescence-associated secretory phenotype (SASP). As senescent cells accumulate in aging tissues, sites of tissue injury, or in response to drugs, SASP factors may contribute to increase platelet activity and, through this mechanism, generate a microenvironment that facilitates cancer progression. Using in vitro models of drug-induced senescence, in which cellular senescence was induced following exposure of mammary epithelial cells (MCF-10A and MCF-7) and gastric cancer cells (AGS) to the CDK4/6 inhibitor Palbociclib, we show that senescent mammary and gastric cells display unique expression profiles of selected SASP factors, most of them being downregulated at the RNA level in senescent AGS cells. In addition, we observed cell-type specific differences in the levels of secreted factors, including IL-1ß, in media conditioned by senescent cells. Interestingly, only media conditioned by senescent MCF-10A and MCF-7 cells were able to enhance platelet aggregation, although all three types of senescent cells were able to attract platelets in vitro. Nevertheless, the effects of factors secreted by senescent cells and platelets on the migration and invasion of non-senescent cells are complex. Overall, platelets have prominent effects on migration, while factors secreted by senescent cells tend to promote invasion. These differential responses likely reflect differences in the specific arrays of secreted senescence-associated factors, specific factors released by platelets upon activation, and the susceptibility of target cells to respond to these agents.

Effect of sex steroid hormone fluctuations in the pathophysiology of male-retinal pigment epithelial cells.

Gender-based differences may influence the occurrence of several ocular conditions suggesting the possibility that fluctuations in sex steroid homeostasis may have direct effects on the eye physiology. Here, we evaluated the effect of sex steroid hormone fluctuations in male retinal pigment epithelial cells, RPEs (ARPE-19). To mimic hormonal fluctuations occurring during aging, we exposed ARPE-19 to acute, prolonged or chronic estradiol, and progesterone challenges. We found that chronic estradiol treatment promotes a remarkable necrosis of RPE cells, and does not affect pRb2/p130 or PAI-2 sub-cellular localization. In contrast, chronic progesterone exposure induces nuclear subcellular rearrangement of pRb2/p130, co-immunolocalization of pRb2/p130 with PAI-2, and accumulation of cells in G2/M phase, which is accompanied by a remarkable reduction of necrosis in favour of apoptosis activation. This study has a high clinical significance since it considers sex steroid fluctuations as inducers of milieu change in the retina able to influence pathological situations occurring with aging in non-reproductive systems such as the eye. Exogenous administration of physiologically significant amounts of sex hormones for long periods of time is a common clinical practice for transgender patients seeking sex reassignment. In particular, our study offers the unique opportunity to unravel the effects of sex hormones, not only in determining gender differences but also in affecting the physiology of non-reproductive systems, such as the eye, in the underserved transgender community.

SERPINs shelter the endowed migrants in a hostile land.

Since metastatic lesions of solid tumors are the major cause of mortality in cancer patients, understanding the molecular mechanisms of metastasis is of paramount importance. Although extensive knowledge has been accumulated regarding the early steps in metastasis--starting with the departure of cancer cells from their primary sites, to their transit through the hematogenous and/or lymphatic systems, and ending with their entrance into the parenchyma of distant organs--it is difficult if not impossible to translate such knowledge into medicine due to the challenge of identifying patients with only primary tumors but otherwise pristine organs. In other words, autopsy studies indicate that a large proportion of patients already harbor dormant, undetectable micrometastases at the time of cancer diagnosis (Hensel et al, 2013). Accordingly, stopping tumor cell dissemination is too late for these patients. Therefore, understanding the survival and outgrowth of micrometastases may hold greater promise to combat metastatic disease.

The role of SerpinB2 in human bronchial epithelial cells responses to particulate matter exposure.

Exposure to particulate matter (PM) has been related to the onset of adverse health effects including lung cancer, but the underlying molecular mechanisms are still under investigation. Epithelial-to-mesenchymal transition (EMT) is regarded as a crucial step in cancer progression. In a previous study, we reported EMT-related responses in the human bronchial epithelial cell line HBEC3-KT, exposed to Milan airborne winter PM2.5. We also found a strong modulation of SERPINB2, encoding for the PAI-2 protein and previously suggested to play an important role in cancer. Here we investigate the role of SERPINB2/PAI-2 in the regulation of EMT-related effects induced by PM exposure in HBEC3-KT. PM exposure (up to 10 µg/cm2) increased SERPINB2 expression, reduced cell migration and induced morphological alterations in HBEC3-KT. Changes in actin structure and cadherin-1 relocalization were observed in PM-exposed samples. Knockdown of SERPINB2 by siRNA down-regulated the CDH1 gene expression, as well as PAI-2 and cadherin-1 protein expression. SERPINB2 knockdown also increased cell migration rate, and counteracted the PM-induced reduction of cell migration and alteration of cell morphology. SERPINB2 was found to be greatly down-regulated in a HBEC2-KT transformed cell line, supporting the importance of this gene in the regulation of EMT. In conclusion, here we show that PAI-2 regulates CDH1 gene/cadherin-1 protein expression in bronchial HBEC3-KT cells, and this mechanism might be involved in the regulation of cell migration. SERPINB2 down-regulation should be considered part of EMT, and the over-expression of SERPINB2 in PM-exposed samples might be interpreted as an initial protective mechanism.

SerpinB2 Deficiency Results in a Stratum Corneum Defect and Increased Sensitivity to Topically Applied Inflammatory Agents.

SerpinB2 (plasminogen activator inhibitor type 2) is constitutively expressed at high levels by differentiating keratinocytes in mice and humans; however, the physiological function of keratinocyte SerpinB2 remains unclear. Herein, we show that SerpinB2(-/-) mice are more susceptible to contact dermatitis after topical application of dinitrofluorobenzene, and show enhanced inflammatory lesions after topical applications of phorbol ester. Untreated SerpinB2(-/-) mice showed no overt changes in epithelial structure, and we were unable to find evidence for a role for keratinocyte SerpinB2 in regulating immunity, apoptosis, IL-1ß production, proteasomal activity, or wound healing. Instead, the phenotype was associated with impaired skin barrier function and a defective stratum corneum, with SerpinB2(-/-) mice showing increased transepidermal water loss, increased overt loss of stratum corneum in inflammatory lesions, and impaired stratum corneum thickening after phorbol ester treatment. Immunoblotting suggested that SerpinB2 (cross-linked into the cornified envelope) is present in the stratum corneum and retains the ability to form covalent inhibitory complexes with urokinase. Data suggest that the function of keratinocyte SerpinB2 is protection of the stratum corneum from proteolysis via inhibition of urokinase, thereby maintaining the integrity and barrier function of the stratum corneum, particularly during times of skin inflammation. Implications for studies involving genetically modified mice treated with topical agents and human dermatological conditions, such as contact dermatitis, are discussed.

Dual-specificity phosphatase 5 regulates nuclear ERK activity and suppresses skin cancer by inhibiting mutant Harvey-Ras (HRasQ61L)-driven SerpinB2 expression.

Ectopic expression of dual-specificity phosphatase 5 (DUSP5), an inducible mitogen-activated protein (MAP) kinase phosphatase, specifically inactivates and anchors extracellular signal-regulated kinase (ERK)1/2 in the nucleus. However, the role of endogenous DUSP5 in regulating the outcome of Ras/ERK kinase signaling under normal and pathological conditions is unknown. Here we report that mice lacking DUSP5 show a greatly increased sensitivity to mutant Harvey-Ras (HRas(Q61L))-driven papilloma formation in the 7,12-Dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) model of skin carcinogenesis. Furthermore, mouse embryo fibroblasts (MEFs) from DUSP5(-/-) mice show increased levels of nuclear phospho-ERK immediately after TPA stimulation and fail to accumulate total ERK in the nucleus compared with DUSP5(+/+) cells. Surprisingly, a microarray analysis reveals that only a small number of Ras/ERK-dependent TPA-responsive transcripts are up-regulated on deletion of DUSP5 in MEFs and mouse skin. The most up-regulated gene on DUSP5 loss encodes SerpinB2, an inhibitor of extracellular urokinase plasminogen activator and deletion of DUSP5 acts synergistically with mutant HRas(Q61L) and TPA to activate ERK-dependent SerpinB2 expression at the transcriptional level. SerpinB2 has previously been implicated as a mediator of DMBA/TPA-induced skin carcinogenesis. By analyzing DUSP5(-/-), SerpinB2(-/-) double knockout mice, we demonstrate that deletion of SerpinB2 abrogates the increased sensitivity to papilloma formation seen on DUSP5 deletion. We conclude that DUSP5 performs a key nonredundant role in regulating nuclear ERK activation, localization, and gene expression. Furthermore, our results suggest an in vivo role for DUSP5 as a tumor suppressor by modulating the oncogenic potential of activated Ras in the epidermis.

The sGC activator inhibits the proliferation and migration, promotes the apoptosis of human pulmonary arterial smooth muscle cells via the up regulation of plasminogen activator inhibitor-2.

BACKGROUND: Different types of pulmonary hypertension (PH) share the same process of pulmonary vascular remodeling, the molecular mechanism of which is not entirely clarified by far. The abnormal biological behaviors of pulmonary arterial smooth muscle cells (PASMCs) play an important role in this process. OBJECTIVES: We investigated the regulation of plasminogen activator inhibitor-2 (PAI-2) by the sGC activator, and explored the effect of PAI-2 on PASMCs proliferation, apoptosis and migration. METHODS: After the transfection with PAI-2 overexpression vector and specific siRNAs or treatment with BAY 41-2272 (an activator of sGC), the mRNA and protein levels of PAI-2 in cultured human PASMCs were detected, and the proliferation, apoptosis and migration of PASMCs were investigated. RESULTS: BAY 41-2272 up regulated the endogenous PAI-2 in PASMCs, on the mRNA and protein level. In PAI-2 overexpression group, the proliferation and migration of PASMCs were inhibited significantly, and the apoptosis of PASMCs was increased. In contrast, PAI-2 knockdown with siRNA increased PASMCs proliferation and migration, inhibited the apoptosis. CONCLUSIONS: PAI-2 overexpression inhibits the proliferation and migration and promotes the apoptosis of human PASMCs. Therefore, sGC activator might alleviate or reverse vascular remodeling in PH through the up-regulation of PAI-2.

TLR-induced PAI-2 expression suppresses IL-1ß processing via increasing autophagy and NLRP3 degradation.

The NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome, a multiprotein complex, triggers caspase-1 activation and maturation of the proinflammatory cytokines IL-1ß and IL-18 upon sensing a wide range of pathogen- and damage-associated molecules. Dysregulation of NLRP3 inflammasome activity contributes to the pathogenesis of many diseases, but its regulation remains poorly defined. Here we show that depletion of plasminogen activator inhibitor type 2 (PAI-2), a serine protease inhibitor, resulted in NLRP3- and ASC (apoptosis-associated Speck-like protein containing a C-terminal caspase recruitment domain)-dependent caspase-1 activation and IL-1ß secretion in macrophages upon Toll-like receptor 2 (TLR2) and TLR4 engagement. TLR2 or TLR4 agonist induced PAI-2 expression, which subsequently stabilized the autophagic protein Beclin 1 to promote autophagy, resulting in decreases in mitochondrial reactive oxygen species, NLRP3 protein level, and pro-IL-1ß processing. Likewise, overexpressing Beclin 1 in PAI-2-deficient cells rescued the suppression of NLRP3 activation in response to LPS. Together, our data identify a tier of TLR signaling in controlling NLRP3 inflammasome activation and reveal a cell-autonomous mechanism which inversely regulates TLR- or Escherichia coli-induced mitochondrial dysfunction, oxidative stress, and IL-1ß-driven inflammation.

[Expression of PAI-2 mRNA in peripheral blood leucocytes and regulation by sGC activator in pulmonary hypertension].

OBJECTIVE: To investigate the mRNA expression level of plasminogen activator inhibitor-2 (PAI-2) in peripheral blood leucocytes and regulation by soluble guanylate cyclase (sGC) activator in pulmonary hypertension. METHODS: The human pulmonary arterial smooth muscle cells were treated with sGC activator Cinaciguat. The mRNA and protein expression levels of PAI-2 were detected with Real-time PCR and Western blot. The fresh blood samples of 8 patients with pulmonary arterial hypertension (PAH) (collected at the China-Japan Friendship Hospital from November 2014 to March 2015), 16 patients with chronic thromboembolic pulmonary hypertension (CTEPH) (collected at the China-Japan Friendship Hospital from November 2014 to March 2015), 24 age- and gender- matched healthy controls (collected at Beijing Hospital in March 2015) were treated with Cinaciguat for 8 hours. Then RNA of peripheral leukocytes was extracted and performed with reverse transcription and Real-time PCR to detect the mRNA level of PAI-2, which was compared between healthy controls and patients with pulmonary hypertension, before and after the treatment of Cinaciguat. At last, the correlation of PAI-2 mRNA level and the clinic severity of pulmonary hypertension were identified. RESULTS: The mRNA and protein expression levels of PAI-2 were promoted by Cinaciguat in human pulmonary arterial smooth muscle cells. The baseline mRNA level of PAI-2 in peripheral leukocytes was significantly lower in PAH patients compared to the healthy controls (0.201±0.152, 0.660±0.440, P=0.021). There was no significant difference in the mRNA expression level of PAI-2 between the CTEPH patients and controls (0.428±0.364, 0.769±0.682, P=0.152). After Cinaciguat treatment, the mRNA expression levels of PAI-2 were up-regulated in PAH patients and CTEPH patients (1.352±1.127, 1.203±1.008), there was no significant difference in the mRNA expression level of PAI-2 among the PAH patients, CTEPH patients and controls (P=0.130, P=0.534). The baseline mRNA level of PAI-2 was negative correlated with echocardiography-estimated systolic pulmonary arterial pressure (sPAP) (r=-0.744, P=0.034). CONCLUSIONS: The mRNA expression level of PAI-2 is significantly down-regulated in peripheral blood leucocytes and up-regulated by sGC activator in pulmonary hypertension. PAI-2 could be used as potential biomarker of pulmonary vascular remodeling in PAH.