LIF, a mitogen for choroidal endothelial cells, protects the choriocapillaris: implications for prevention of geographic atrophy
2021; Springer Nature; Volume: 14; Issue: 1 Linguagem: Inglês
10.15252/emmm.202114511
ISSN1757-4684
AutoresPin Li, Qin Li, Nilima Biswas, Hong Xin, Tanja Diemer, Lixian Liu, Lorena Pérez, Giovanni Paternostro, Carlo Piermarocchi, Sergii Domanskyi, Ruikang K. Wang, Napoleone Ferrara,
Tópico(s)Neutrophil, Myeloperoxidase and Oxidative Mechanisms
ResumoArticle15 November 2021Open Access Source DataTransparent process LIF, a mitogen for choroidal endothelial cells, protects the choriocapillaris: implications for prevention of geographic atrophy Pin Li Pin Li Department of Pathology, University of California San Diego, La Jolla, CA, USA These authors contributed equally to this work as first authors Search for more papers by this author Qin Li Qin Li orcid.org/0000-0001-6286-1023 Department of Ophthalmology, University of California San Diego, La Jolla, CA, USA These authors contributed equally to this work as first authors Present address: BioDuro-Sundia, Inc., San Diego, CA, USA. Search for more papers by this author Nilima Biswas Nilima Biswas Department of Pathology, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Hong Xin Hong Xin Department of Pathology, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Tanja Diemer Tanja Diemer Department of Pathology, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Lixian Liu Lixian Liu Department of Pathology, University of California San Diego, La Jolla, CA, USA Present address: Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Lorena Perez Gutierrez Lorena Perez Gutierrez Department of Pathology, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Giovanni Paternostro Giovanni Paternostro orcid.org/0000-0003-4029-5467 Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA Search for more papers by this author Carlo Piermarocchi Carlo Piermarocchi Department of Physics and Astronomy, Michigan State University, East Lansing, MI, USA Search for more papers by this author Sergii Domanskyi Sergii Domanskyi orcid.org/0000-0002-6847-6019 Department of Physics and Astronomy, Michigan State University, East Lansing, MI, USA Search for more papers by this author Ruikang K Wang Ruikang K Wang orcid.org/0000-0001-5169-8822 Department of Bioengineering, University of Washington, Seattle, WA, USA Search for more papers by this author Napoleone Ferrara Corresponding Author Napoleone Ferrara [email protected] orcid.org/0000-0002-7029-6375 Department of Pathology, University of California San Diego, La Jolla, CA, USA Department of Ophthalmology, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Pin Li Pin Li Department of Pathology, University of California San Diego, La Jolla, CA, USA These authors contributed equally to this work as first authors Search for more papers by this author Qin Li Qin Li orcid.org/0000-0001-6286-1023 Department of Ophthalmology, University of California San Diego, La Jolla, CA, USA These authors contributed equally to this work as first authors Present address: BioDuro-Sundia, Inc., San Diego, CA, USA. Search for more papers by this author Nilima Biswas Nilima Biswas Department of Pathology, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Hong Xin Hong Xin Department of Pathology, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Tanja Diemer Tanja Diemer Department of Pathology, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Lixian Liu Lixian Liu Department of Pathology, University of California San Diego, La Jolla, CA, USA Present address: Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Lorena Perez Gutierrez Lorena Perez Gutierrez Department of Pathology, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Giovanni Paternostro Giovanni Paternostro orcid.org/0000-0003-4029-5467 Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA Search for more papers by this author Carlo Piermarocchi Carlo Piermarocchi Department of Physics and Astronomy, Michigan State University, East Lansing, MI, USA Search for more papers by this author Sergii Domanskyi Sergii Domanskyi orcid.org/0000-0002-6847-6019 Department of Physics and Astronomy, Michigan State University, East Lansing, MI, USA Search for more papers by this author Ruikang K Wang Ruikang K Wang orcid.org/0000-0001-5169-8822 Department of Bioengineering, University of Washington, Seattle, WA, USA Search for more papers by this author Napoleone Ferrara Corresponding Author Napoleone Ferrara [email protected] orcid.org/0000-0002-7029-6375 Department of Pathology, University of California San Diego, La Jolla, CA, USA Department of Ophthalmology, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Author Information Pin Li1, Qin Li2, Nilima Biswas1, Hong Xin1, Tanja Diemer1, Lixian Liu1, Lorena Perez Gutierrez1, Giovanni Paternostro3, Carlo Piermarocchi4, Sergii Domanskyi4, Ruikang K Wang5 and Napoleone Ferrara *,1,2 1Department of Pathology, University of California San Diego, La Jolla, CA, USA 2Department of Ophthalmology, University of California San Diego, La Jolla, CA, USA 3Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA 4Department of Physics and Astronomy, Michigan State University, East Lansing, MI, USA 5Department of Bioengineering, University of Washington, Seattle, WA, USA *Corresponding author. Tel: +858 822 6822; E-mail: [email protected] EMBO Mol Med (2021)e14511https://doi.org/10.15252/emmm.202114511 Present address: BioDuro-Sundia, Inc., San Diego, CA, USA. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract In the course of our studies aiming to discover vascular bed-specific endothelial cell (EC) mitogens, we identified leukemia inhibitory factor (LIF) as a mitogen for bovine choroidal EC (BCE), although LIF has been mainly characterized as an EC growth inhibitor and an anti-angiogenic molecule. LIF stimulated growth of BCE while it inhibited, as previously reported, bovine aortic EC (BAE) growth. The JAK-STAT3 pathway mediated LIF actions in both BCE and BAE cells, but a caspase-independent proapoptotic signal mediated by cathepsins was triggered in BAE but not in BCE. LIF administration directly promoted activation of STAT3 and increased blood vessel density in mouse eyes. LIF also had protective effects on the choriocapillaris in a model of oxidative retinal injury. Analysis of available single-cell transcriptomic datasets shows strong expression of the specific LIF receptor in mouse and human choroidal EC. Our data suggest that LIF administration may be an innovative approach to prevent atrophy associated with AMD, through protection of the choriocapillaris. Synopsis Little progress has been made in the therapy of the dry form of age-related macular degeneration (AMD). We identified LIF as a novel mitogen and survival factor for choroidal EC. Protection of the choriocapillaris may be an innovative approach to prevent atrophy associated with AMD. LIF, a member of the IL-6 family, is identified as a mitogen for cultured bovine choroidal ECs. LIF induces retinal and choroidal angiogenesis. LIF has protective effects on the choriocapillaris in a model of oxidative injury, suggesting that it may help prevent the atrophy associated with AMD. The paper explained Problem Major progress has been made in the prevention of blindness secondary to intraocular vascular diseases such as neovascular age-related macular degeneration (AMD) through the use of VEGF inhibitors. However, dry AMD and geographic atrophy (GA) are major causes of vision loss in the elderly populations, and there are currently no effective treatments for these diseases. Loss of choriocapillaris (CC) has been shown to be an early event in AMD that precedes degeneration of retinal pigment epithelium (RPE). We hypothesized that strategies aiming to protect and/or regenerate the CC, potentially together with the RPE, may have therapeutic value for GA. Results Using a biochemical/functional approach, we sought to identify novel factors that may have protective effects on choroidal endothelial cells (CEC). We unexpectedly identified LIF, a member of the IL-6 family, as a mitogen for cultured bovine CEC, although LIF has been previously characterized as an EC growth inhibitor. We show that LIF stimulation of the JAK-STAT3 pathway mediates, depending on the EC type, both growth stimulation and growth inhibition. We also discovered that growth inhibition is mediated by STAT3-induced cathepsin-dependent apoptotic cell death and cell cycle arrest. Our data also show that LIF induces retinal and choroidal angiogenesis following intravitreal administration in the mouse. In addition, LIF and the related IL-6 family member cardiotrophin-1 (CT-1) had protective effects on the CC in a model of oxidative injury, suggesting that these agents may help prevent the atrophy associated with AMD. Analysis of available single-cell transcriptomic datasets shows high expression of the specific LIF receptor (LIFR) in human choroidal EC, comparable to the VEGF receptors. Impact This study advances our understanding of EC diversity and sheds light on EC-type specificity of STAT3 signaling, potentially explaining some paradoxical results. Our data further suggest LIF administration as an innovative approach to prevent atrophy associated with AMD, through protection of the CC. Introduction Angiogenesis is a major developmental and physiological process (Yancopoulos et al, 2000; Chung et al, 2010; Chung & Ferrara, 2011). The growth of new blood vessels is also a key aspect of a variety of pathological conditions, including tumors and intraocular vascular disorders (Adams & Alitalo, 2007; Chung & Ferrara, 2011). The newly formed vessels provide growing tumors with nutrients and oxygen and thus play an important role in tumor progression. Also, growth of abnormal and leaky blood vessels is associated with a variety of blinding ocular disorders (Adams & Alitalo, 2007; Chung & Ferrara, 2011; Potente et al, 2011). Over the last few decades, extensive efforts have been made to dissect the molecular basis of angiogenesis and to identify potential therapeutic targets. These efforts resulted in the discovery of the major signaling pathways involved in normal and abnormal vascular development (Adams & Alitalo, 2007; Chung & Ferrara, 2011; Potente et al, 2011). Much research has established the key role of the VEGF pathway in normal and pathological angiogenesis (Ferrara & Adamis, 2016; Apte et al, 2019). Indeed, current therapeutic approaches in angiogenesis rely on inhibiting the VEGF/VEGFR or Ang2/Tie2 pathways. Conversely, promoting collateral vessel growth could provide a clinical benefit to patients with ischemic disorders, who have limited pharmacological options (Ferrara & Alitalo, 1999). This hypothesis led to numerous clinical trials in the past decades, testing a variety of angiogenic factors (e.g., VEGF-A, VEGF-C, HGF, FGF-1, FGF-2, and FGF-4), in patients with coronary or limb ischemia (Isner, 1996; Carmeliet, 2005; Ferrara & Kerbel, 2005). However, these studies did not demonstrate therapeutic benefits, in spite of encouraging results in animal models (Simons, 2005). Thus, there is a need to further elucidate the molecular and biological basis of therapeutic angiogenesis. Interestingly, earlier studies suggested the possibility of organ-specific regulation of angiogenesis. Our laboratory described EG-VEGF, a mitogen with a selectivity for endocrine gland EC (LeCouter et al, 2001). This finding raised the possibility that other factors with a selectivity for EC of specific vascular beds may exist. Indeed, several studies have emphasized the importance of organotypic properties of EC, e.g., the vascular bed-specific release of growth factors and cytokines that can stimulate organ-specific growth and regeneration (LeCouter et al, 2003; Red-Horse et al, 2007; Rafii et al, 2016; Augustin & Koh, 2017). Age-related macular degeneration (AMD) is a leading cause of vision loss in Americans 50 years and older (Vingerling et al, 1995; Jager et al, 2008). Both genetic and environmental factors contribute to the pathogenesis of AMD (Jager et al, 2008; Zhang et al, 2012; Ambati et al, 2013; Yang et al, 2006). Early-stage AMD is characterized by drusen and abnormalities of the retinal pigment epithelium (RPE). Late-stage AMD can be neovascular (nv) (also known as wet or exudative) or non-neovascular (known as atrophic or dry) (Ferris et al, 1984; Jager et al, 2008; Mitchell et al, 2018). While 10–15% of patients with intermediate AMD progress to nv form, the remaining patients who progress develop geographic atrophy (GA) (Jager et al, 2008; Fleckenstein et al, 2018). GA is characterized by severe visual impairment and scotomas due to loss of photoreceptors, RPE, and choriocapillaris (CC). The last decade has witnessed significant progress in the prevention of blindness secondary to nv AMD (Ferrara & Adamis, 2016). Unfortunately, little progress has been possible in the therapy of dry AMD and GA. Loss of CC has been documented by histopathology and, more recently, through optical coherence tomography–angiography (OCT-A) as an early event in AMD, and has been reported to occur underneath and beyond the areas of photoreceptor and RPE loss (Mullins et al, 2011b; Moreira-Neto et al, 2018). Indeed, loss of CC has been reported to precede RPE degeneration (Curcio et al, 2000; Biesemeier et al, 2014). Recent studies provide evidence for deposition of membrane attack complexes in the choroid of patients with high-risk CFH genotype (Mullins et al, 2011a). Therefore, loss of CC is strongly implicated in the pathogenesis of dry AMD and GA (Arya et al, 2018; Moreira-Neto et al, 2018), raising the possibility that strategies aiming at protecting and/or regenerating the CC, together with the RPE, may be valuable. VEGF probably would not be suitable in this setting, given its vascular permeability-enhancing properties (Apte et al, 2019). Intriguingly, a recent exploratory analysis from the AREDS2 study has reported that enlargement of recent GA lesions slows down before the onset of nv AMD lesions, suggesting that non-exudative nv tissue may prevent GA progression, likely due to perfusion improvement (Hwang et al, 2021). In search of novel regulators of choroidal EC growth/survival, we screened human glioblastoma (GBM) cell lines since tumors have been historically proven to be a rich source of angiogenic factors (Kerbel, 2000). To identify such factors, we employed an unbiased proteomic/functional approach that relies on stimulating growth of primary bovine choroidal EC (BCE). We found that a particular GBM cell line (LN-229) had unusual features that distinguished it from typical GBM, i.e., it had a very low VEGF expression (Depner et al, 2016). Intriguingly, the LN-229-conditioned medium had mitogenic effects on BCE, although none of the conventional EC mitogens was detectable. We identified LIF, a member of the IL-6 family (Murakami et al, 2019), as the mitogen, although LIF has been previously characterized as an EC growth inhibitor. To understand the basis of such paradoxical effects, we analyzed signaling events elicited by LIF in BCE and bovine aortic EC (BAE), the EC type that was originally reported to be growth inhibited by LIF (Ferrara et al, 1992; Takashima & Klagsbrun, 1996). We also sought to establish whether LIF induces angiogenesis when injected in the eye and explored the possibility that LIF may have protective effects in models of injury to the CC, a structure that is critically affected in AMD. Results Identification of LIF as a mitogen for choroidal EC Media conditioned by LN-229 cells (LN-229 CM) stimulated growth of bovine choroidal EC (BCE) (Fig 1A). However, in agreement with previous studies (Depner et al, 2016), LN-229 cells secreted very little VEGF in the medium (Appendix Fig S1A). The anti-VEGF antibody B20-4.1 did not suppress the mitogenic effects of LN-229 CM (Fig 1B), suggesting the involvement of VEGF-independent pathways. We examined the angiogenic factors profile of LN-229 CM using specific antibody arrays. This analysis revealed that the majority of potential EC mitogens, including FGF-1, FGF-2, or HGF, were undetectable, except PDGF-AA, CCL2 (also known as MCP-1), and interleukin 8 (IL-8), which were abundant in the CM (Appendix Fig S1B). However, antibodies neutralizing PDGF-AA or CCL2 failed to suppress BCE cell growth induced by the LN-229 CM (Appendix Fig S1C and D). In pilot experiments, we determined that IL-8 lacks mitogenic effects on BCE cells. Figure 1. Identification of LIF as the EC mitogen in LN-229-conditioned medium (CM) LN-229 CM stimulated growth of BCE cells, n = 3. VEGF neutralizing antibody (B20) failed to suppress BCE cell growth induced by LN-229 CM, n = 3. Reverse-phase fractions of LN-229 CM induced BCE cell growth. BCE cells were incubated with fractions (2 μl/well) as indicated in the figure, n = 3. Candidate proteins generated from mass-spectrometry analysis of LN-229 CM reverse-phase fractions. Candidates were identified by excluding intracellular proteins and proteins showing higher abundance in inactive fractions compared to those in mitogenic factions. Proteins were ranked for relative abundance as described in Materials and Methods. The anti-LIF neutralizing antibody abolished BCE cell growth induced by reverse-phase fractions, n = 3. Recombinant human LIF proteins stimulated growth of BCE cells in a dose-dependent manner. BCE cells were cultured in the presence of vehicle, VEGF (10 ng/ml), and the indicated concentrations of recombinant human LIF (rhLIF, Sigma), n = 3. LIF and VEGF synergistically stimulated BCE cell growth. Cell proliferation was analyzed after 6 days using alamar blue as described in Materials and Methods, n = 3. Data information: Bars and error bars represent mean ± SD. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test. ns, not statistically significant. Source data are available online for this figure. Source Data for Figure 1 [emmm202114511-sup-0007-SDataFig1.xlsx] Download figure Download PowerPoint To identify BCE mitogens in the LN-229 CM, we undertook a biochemical/functional approach. The BCE mitogenic activity was enriched through two sequential chromatographic steps, anion-exchange and reverse-phase chromatography. At each step, only one peak of absorbance, composed of 4–5 contiguous fractions, showed mitogenic activity. The reverse-phase column fractions were labeled as R1-R45. The peak mitogenic fractions (R26 and R27), the minimally mitogenic (R25 and R28), and adjacent negative (R24 and R29) fractions (Fig 1C) were subjected to mass spectrometry analyses. A short list of five candidate proteins was generated by screening out intracellular proteins (Fig 1D). Four of the five proteins were serum components and/or redox enzymes (i.e., peroxiredoxins and alpha-2-macroglobulin), while LIF stood out as a cytokine. LIF, a member of the interleukin 6 (IL-6) family of proteins (Murakami et al, 2019), is broadly expressed and exerts effects in multiple cell types and tissues, and has been implicated in various critical physiological processes including embryonic stem cell self-renewal and blastocyst implantation (Nicola & Babon, 2015). The presence of LIF herein was unexpected, since this cytokine had been previously characterized as an EC growth inhibitor and an anti-angiogenic agent (Pepper et al, 1995; Ash et al, 2005; Kubota et al, 2008). Nevertheless, an anti-LIF polyclonal antibody completely suppressed growth of BCE cells induced by the reverse-phase fractions (Fig 1E). These observations suggested that LIF might be responsible for the mitogenic effects. Indeed, recombinant LIF stimulated growth of BCE cells (Fig 1F) and bovine retinal EC (BRE) cells (Appendix Fig S2A). Interestingly, VEGF and LIF resulted in more than additive mitogenic effects in both BCE (Fig 1G) and BRE cells (Appendix Fig S2B), suggesting a synergistic relationship between the two factors. In addition, we tested the mitogenic effect of LIF on several human ECs. LIF stimulated the proliferation of human choroidal EC and human liver sinusoidal ECs to an extent comparable with VEGF (Appendix Fig S2C and D). Stimulation of BCE growth is mediated by the JAK-STAT3 pathway Although all members of the IL-6 family share a receptor component, gp130, LIF signaling transduces via the gp130:LIFR receptor dimer, while IL-6 activates its downstream signal through the IL6Rα:gp130:gp130:IL6Rα tetramer (Nicola & Babon, 2015; Murakami et al, 2019). Among four Janus kinases (JAK1, JAK2, JAK3, and TYK2) associated with gp130, LIF signaling selectively activates JAK1 through transphosphorylation (Rodig et al, 1998; Nicola & Babon, 2015). Upon activation by LIF, JAKs elicit three distinct signaling cascades: JAK-STAT, PI3K-AKT-mTOR, and RAS-MAPK, which contribute to different functions in a cell-type-specific manner. As to JAK-STAT pathway, LIF signaling preferentially activates STAT3, although STAT1 and STAT5 can also be phosphorylated by JAK1 (Kiu & Nicholson, 2012). To examine which pathways are responsible for LIF-induced growth stimulation in BCE cells, we employed a set of small-molecule inhibitors: baricitinib, cobimetinib, and BEZ235, which are specifically against JAK1/2, MEK1/2(MAPK pathway), and PI3K/mTOR, respectively (Serra et al, 2008; Liu et al, 2019; Tong et al, 2020). In BCE cells, LIF treatment for 15 min elicited phosphorylation of STAT3 and ERK but had little effect on AKT phosphorylation (Fig 2A). Preincubation with the JAK1/2 inhibitor baricitinib almost completely suppressed LIF-induced STAT3 and ERK/MAPK phosphorylation (Fig 2A), while cobimetinib pretreatment blocked ERK phosphorylation but showed no effects on STAT3 and AKT phosphorylation (Fig 2A). BEZ235 had only moderate effects on AKT phosphorylation regardless of LIF treatment (Fig 2A). Moreover, baricitinib completely blocked LIF-induced cell growth, while cobimetinib showed minimal effects and the PI3K/mTOR inhibitor BEZ235 had no effect on LIF-stimulated cell growth (Fig 2B). These observations suggested that the MAPK and PI3K pathways might not be major contributors to LIF stimulation in BCE cells, and thus JAK-STAT might be implicated. Since STAT3 is the preferential mediator in LIF-induced JAK-STAT signaling cascade (Kiu & Nicholson, 2012) and has been implicated in proliferation and survival in a wide variety of cell types, we further examined the role of STAT3 in BCE by siRNA knockdown. siRNAs successfully dampened STAT3 levels at both RNA and protein levels in BCE cells (Fig 2C and D). Downregulation of STAT3 blocked LIF-induced BCE cell growth in vitro (Fig 2E). Moreover, LIFR siRNAs abolished the growth-promoting effect of LIF in BCE cells, confirming that the proliferation was mediated via the LIF/LIFR pathway (Appendix Fig S3A and B). Figure 2. LIF promotes BCE cell growth via the JAK-STAT3 pathway A. The JAK inhibitor baricitinib (Ba) blocked LIF-induced STAT3 phosphorylation. BCE cells were preincubated with DMSO, baricitinib (2 μM), cobimetinib (Co) (150 nM), or BEZ235 (BE) (5 nM) for 1 h and were then treated with vehicle or LIF (10 ng/ml, Sigma) for 15 min. Ctrl, no preincubation with inhibitors. B. Baricitinib suppressed LIF-induced BCE cell growth. BCE cells were preincubated with DMSO, baricitinib, cobimetinib, or BEZ235 for 1 h and then treated with vehicle, LIF (10 ng/ml), or VEGF (10 ng/ml). Cell proliferation was analyzed after 6 days, n = 3. C, D. STAT3 knockdown in BCE cells. BCE cells were transfected with siNegative and siRNAs targeting STAT3. qRT-PCR was performed to examine STAT3 mRNA levels. STAT3 level in siNegative was set as 1. Data from three independent experiments were averaged and are presented in C. In D, cells transfected with siRNAs were treated with LIF (10 ng/ml) or vehicle for 15 min. Whole-cell lysates were subjected to Western blotting with the indicated antibodies. E. LIF-induced BCE cell growth was abolished by STAT3 knockdown. BCE cells with STAT3 knockdown were cultured with LIF (10 ng/ml, Sigma) or vehicle. Cell proliferation was analyzed after 3 days. Fluorescence reading at 590 nm for each vehicle group was set as 1, n = 3. siNegative, negative control siRNA not targeting any known genes. Data information: Bars and error bars represent mean ± SD. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test. Source data are available online for this figure. Source Data for Figure 2 [emmm202114511-sup-0008-SDataFig2.zip] Download figure Download PowerPoint LIF inhibits BAE growth via the JAK-STAT3 pathway In agreement with previous studies (Ferrara et al, 1992), LIF resulted in BAE cell growth inhibition (Fig 3A), To interrogate the LIF-induced signaling cascade in BAE cells, we again used baricitinib, cobimetinib, and BEZ235 in order to inhibit LIF-gp130:LIFR downstream components JAK1/2, MEK1/2, and PI3K/mTOR (Serra et al, 2008; Liu et al, 2019; Tong et al, 2020). In BAE cells, LIF treatment for 15 min led to phosphorylation of STAT3, ERK (MAPK), and AKT (Fig 3B). Baricitinib pretreatment significantly suppressed LIF-induced phosphorylation of STAT3, ERK, and AKT, while cobimetinib and BEZ235 pretreatment also effectively repressed phosphorylation of ERK and AKT, respectively (Fig 3B). Remarkably, baricitinib was the only inhibitor that reversed growth suppression induced by LIF in BAE cells (Fig 3C), suggesting that the JAK-STAT pathway mediated effects of LIF in BAE cells. To further examine whether inhibition of BAE cells by LIF was attributed to the JAK-STAT3 cascade, STAT3 was knocked down by approximately 80% with three different siRNA in BAE cells (Fig 3D and E). Interestingly, knockdown of STAT3 in BAE cells reduced growth inhibition by LIF (Fig 3F). Consistent with these findings, LIFR knockdown by siRNA also abolished the inhibitory effects of LIF on BAE cells (Appendix Fig S3C and D). These findings further support the notion that LIF inhibited BAE growth via LIF/LIF receptor and JAK-STAT3 pathway. Figure 3. LIF inhibits BAE cell growth through the JAK-STAT3 pathway A. Recombinant human LIF inhibited growth of BAE cells in a dose-dependent manner. BAE cells were cultured in the presence of vehicle and indicated concentrations of recombinant human LIF (rhLIF). Cell proliferation was analyzed after 6 days, n = 3. B. JAK inhibitor baricitinib blocked activation of STAT3 by LIF. BAE cells preincubated with DMSO and inhibitors for 1 h were treated with vehicle and LIF (10 ng/ml) for 15 min. Whole-cell lysates were subjected to Western blotting with indicated antibodies. Ctrl, no preincubation with inhibitors; Ba, baricitinib (2 μM); Co, cobimetinib (150 nM); BE, BEZ235 (5 nM). C. The JAK inhibitor baricitinib reversed LIF-induced BAE growth inhibition. BAE cells preincubated with inhibitors for 1 h were treated with vehicle and LIF (10 ng/ml, Sigma). Cell proliferation was analyzed after 6 days using alamar blue, n = 3. D, E. Knockdown of STAT3 in BAE cells. BAE cells were transfected with siRNAs targeting STAT3. qRT-PCR was performed to examine STAT3 mRNA levels. STAT3 level in siNegative was set as 1. Data from three independent experiments were averaged and shown in D. In E, cells transfected with siRNAs were treated with LIF (10 ng/ml, Sigma) and vehicle for 15 min. Whole-cell lysates were subjected to Western blotting with indicated antibodies. F. LIF-induced BAE cell growth inhibition was abolished by knockdown of STAT3. BAE cells with STAT3 knockdown were cultured with LIF (10 ng/ml, Sigma) and vehicle. Cell proliferation was analyzed after 3 days. Fluorescence reading for each vehicle group was set as 1, n = 3. Data information: Bars and error bars represent mean ± SD. All experiments were carried out in three independent studies. siNegative, negative control siRNA not targeting any known genes. Two-way ANOVA was used as statistical test. Source data are available online for this figure. Source Data for Figure 3 [emmm202114511-sup-0009-SDataFig3.zip] Download figure Download PowerPoint LIF inhibits BAE growth via cathepsin L-dependent cell death and cell cycle arrest We next examined which growth inhibitory effects (e.g., cell cycle arrest, cellular senescence, or programmed cell death) were induced by LIF in BAE cells. Since IL-6-STAT3 signaling was tightly associated with cellular senescence (Kojima et al, 2013), we first hypothesized that LIF-STAT3 axis also induced senescence in BAE cells. However, in the senescence-associated β-galactosidase assay, we did not observe increased numbers of senescent cells in BAE cell treated with LIF for 48 h (Appendix Fig S4A), suggesting that senescence was not the main effect elicited by LIF in BAE cells. Interestingly, staining for the cell death marker Annexin V showed an increased proportion of cells were Annexin V positive in BAE cells treated with LIF for 24 h (Appendix Fig S4B and C), indicating that LIF treatment induced cell death. Surprisingly, co-incubation with the caspase inhibitors (Q-VD-OPH, Z-VAD-fmk, and Z-DEVD-fmk) or poly (adenosine 5'-diphosphate ribose) polymerase (PARP) inhibitor (5-AIQ) did not prevent cell death ind
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