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Vascular endothelial growth factor receptor 2 (VEGFR2) plays crucial roles in vasculogenesis, a process involving cell proliferation, migration and differentiation. However, the molecular mechanism by which VEGFR2 signaling directs vascular endothelial differentiation of VEGFR2+ mesodermal progenitors is not well understood. In this study, we examined the signal transduction pathway downstream of VEGFR2 for endothelial differentiation using an in vitro differentiation system of mouse embryonic stem-cell-derived VEGFR2+ cells. Using chimeric receptors composed of VEGFR2 and VEGFR3, the third member of the VEGFR family, we found that signaling through tyrosine 1175 (Y1175, corresponding to mouse Y1173) of VEGFR2 is crucial for two processes of endothelial differentiation: endothelial specification of VEGFR2+ progenitors, and subsequent survival of endothelial cells (ECs). Furthermore, we found that phospholipase Cγ1 (PLCγ1), which interacts with VEGFR2 through phosphorylated Y1175, is an inducer of endothelial specification. In contrast to VEGFR2, VEGFR3 does not transmit a signal for endothelial differentiation of VEGFR2+ cells. We found that VEGFR3 does not activate PLCγ1, although VEGFR3 has the ability to support endothelial cell survival. Taken together, these findings indicate that VEGFR2-PLCγ1 signal relay gives rise to the unique function of VEGFR2, thus enabling endothelial differentiation from vascular progenitors. [ABSTRACT FROM AUTHOR]

At present, the molecular mechanisms driving the progression and metastasis of oral squamous cell carcinoma (OSCC) remain largely uncharacterized. The activation of transforming growth factor-β (TGF-β) signaling in the tumor microenvironment has been observed in various types of cancer and has been implicated their progression by enhancing the migration and invasion of epithelial cancer cells. However, its specific roles in the oral cancer progression remain unexplored. In this study, we examined the effects of TGF-β signaling on the murine squamous cell carcinoma, SCCVII cells in vitro and in vivo. The incubation of SCCVII cells with TGF-β induced the activation of TGF-β signals and epithelial-mesenchymal transition (EMT). Notably, the motility of SCCVII cells was increased upon the activation of the TGF-β signaling. RNA sequencing revealed upregulation of genes related to EMT and angiogenesis. Consistent with these in vitro results, the inhibition of TGF-β signals in SCCVII cell-derived primary tumors resulted in suppressed angiogenesis. Furthermore, we identified six candidate factors (ANKRD1, CCBE1, FSTL3, uPA, TSP-1 and integrin β3), whose expression was induced by TGF-β in SCCVII cells, and associated with poor prognosis for patients with head and neck squamous cell carcinoma. These results highlight the role of TGF-β signals in the progression of OSCC via multiple mechanisms, including EMT and angiogenesis, and suggest novel therapeutic targets for the treatment of OSCC. • TGF-β enhances the squamous cell carcinoma (SCC) cell migration via induction of EMT. • TGF-β signals induce the expression of genes related to EMT and angiogenesis. • Inhibition of TGF-β signals suppresses angiogenesis in SCC cell-derived primary tumor. • TGF-β upregulates genes associated with poor prognosis for patients with HNSCC. [ABSTRACT FROM AUTHOR]

Prevascularized artificial three-dimensional (3D) tissues are effective biomaterials for regenerative medicine. We have previously established a scaffold-free 3D artificial vascular tissue from normal human dermal fibroblasts (NHDFs) and umbilical vein-derived endothelial cells (HUVECs) by layer-by-layer cell coating technique. In this study, we constructed an artificial vascular tissue constructed by human adipose tissue-derived stromal cells (hASCs) and HUVECs (ASCVT) by a modified technique with cryopreservation. ASCVT showed a higher thickness with more dense vascular networks than the 3D tissue based on NHDFs. Correspondingly, 3D-cultured ASCs showed higher expression of several angiogenesis-related factors, including vascular endothelial growth factor-A and hepatic growth factor, compared to that of NHDFs. Moreover, perivascular cells in ASCVT were detected by pericyte markers, suggesting the differentiation of hASCs into pericyte-like cells. Subcutaneous transplantation of ASCVTs to nude mice resulted in an engraftment with anastomosis of host's vascular structures at 2 weeks after operation. In the engrafted tissue, the vascular network was surrounded by mural-like structure-forming hASCs, in which some parts developed to form vein-like structures at 4 weeks, suggesting the generation of functional vessel networks. These results demonstrated that cryopreserved human cells, including hASCs, could be used directly to construct the artificial transplantable tissue for regenerative medicine. [ABSTRACT FROM AUTHOR]

Presents information on the identification of prostate stem cell antigen (PSCA) importance to the development of diagnostic and therapeutic modalities for the management of prostate cancer. Indentification of the PSCA; Background information on prostates cancer; Information on PSCA expression; Methods used in the identification process.

Lymphangioleiomyomatosis (LAM) is a rare pulmonary disease characterised by the proliferation of smooth muscle-like cells (LAM cells), and an abundance of lymphatic vessels in LAM lesions. Studies reported that vascular endothelial growth factor-D (VEGF-D) secreted by LAM cells contributes to LAM-associated lymphangiogenesis, however, the precise mechanisms of lymphangiogenesis and characteristics of lymphatic endothelial cells (LECs) in LAM lesions have not yet been elucidated. In this study, human primary-cultured LECs were obtained both from LAM-affected lung tissues (LAM-LECs) and normal lung tissues (control LECs) using fluorescence-activated cell sorting (FACS). We found that LAM-LECs had significantly higher ability of proliferation and migration compared to control LECs. VEGF-D significantly promoted migration of LECs but not proliferation of LECs in vitro. cDNA microarray and FACS analysis revealed the expression of vascular endothelial growth factor receptor (VEGFR)-3 and integrin α9 were elevated in LAM-LECs. Inhibition of VEGFR-3 suppressed proliferation and migration of LECs, and blockade of integrin α9 reduced VEGF-D-induced migration of LECs. Our data uncovered the distinct features of LAM-associated LECs, increased proliferation and migration, which may be due to higher expression of VEGFR-3 and integrin α9. Furthermore, we also found VEGF-D/VEGFR-3 and VEGF-D/ integrin α9 signaling play an important role in LAM-associated lymphangiogenesis. [ABSTRACT FROM AUTHOR]

Objective: The in vivo mechanoresponsive and lubricating changes of the mandibular condylar cartilage (MCC) associated with mandibular lateral shift (MLS) and recovery are poorly understood. Using growing rats, we investigated whether the expression of mechanoresponsive factors, including proteoglycan-4 (PRG4), Indian hedgehog (Ihh) and transforming growth factor-β1 (TGF-β1), would be affected by MLS. We also investigated whether these changes could recover to the control level after a 2-week treatment reversal (TR). Materials and methods: The MLS appliances were placed for 2 or 4 weeks in 5-week-old rats and removed from 7-week-old rats in the TR group. The MCC was analysed histomorphometrically by toluidine blue staining. Reverse transcription-polymerase chain reaction and immunohistochemistry were performed to evaluate the expression of PRG4, Ihh, PTHrP (parathyroid hormone-related protein), TGF-β1, Matrix metallopeptidase 13 (MMP-13) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5). Results: A thickened superficial layer and an enhanced expression of PRG4 were detected in MLS groups. PTHrP-Ihh expression correlated positively with the up-regulation of PRG4. TGF-β1 expression decreased in the early stage of MLS but recovered to the control level in the TR group. A significantly enhanced expression of MMP-13 in MLS groups was detected. Conclusion: MLS treatment, which acted on the growth stage of rats, affected the morphology and expression of lubrication factor in the MCC. Elimination of this mechanical stimulus may help MCC recover to normal conditions. Clinical relevance: Our study supports that the adaptive changes of MCC, which are caused by mandibular functional deviation, could be largely recovered by early treatment. [ABSTRACT FROM AUTHOR]

Highlights • Nasal obstruction in neonatal rats induced a decreased of SpO 2 regardless of weight and also affected their taste sensing. • Sweet taste preference was especially decreased among five basic tastes (sweet, salty, sour, bitter, umami). • The quantitative RT-PCR revealed that the mRNA expressions of representative sweet taste-related molecules; T1R2, T1R3, α-gustducin, and PLCβ2 in the circumvallate papillae were decreased in nasal obstruction rats. Abstract Nasal obstruction causes mouth breathing, and affects the growth and development of craniofacial structures, muscle function in the stomatognathic system, and the taste perceptive system. However, the detailed mechanism underlying the effects of nasal obstruction on taste perception has not been fully elucidated. In this study, we investigated this mechanism using the two-bottle taste preference test, immunohistological analysis, and quantification of the mRNA expression of taste-related molecules in the circumvallate papillae. Neonatal male Wistar rats were divided randomly into control and experimental groups. Rats in the experimental group underwent unilateral nasal obstruction by cauterization of the external nostril at the age of 8 days. Arterial oxygen saturation (SpO 2) was recorded in awake rats using collar clip sensors. Taste preference for five basic taste solutions was evaluated. Immunohistochemical analysis and quantitative real-time polymerase chain reaction (RT-PCR) were conducted to evaluate the expressions of taste-related molecules in the taste cells of the circumvallate papillae. Body weights were similar between the two groups throughout the experimental period. The SpO 2 in the 7- to 12-week-old rats in the experimental group was significantly lower than that in the age-matched rats in the control group. In the two-bottle taste preference test, the sensitivities to sweet taste decreased in the experimental group. The mRNA expression of T1R2 , T1R3 , α-gustducin , and PLCβ2 was significantly lower in the experimental group than in the control group as determined by quantitative RT-PCR, and the immunohistochemical staining for α-gustducin and PLCβ2 was less prominent. These findings suggest that nasal obstruction may affect sweet taste perception via the reduced expression of taste-related molecules in the taste cells in rat circumvallate papillae. [ABSTRACT FROM AUTHOR]

Mammalian or mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth, metabolism, and cell differentiation. Recent studies have revealed that the recruitment of mTORC1 to lysosomes is essential for its activation. The ceramide analogue 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a well known glycosphingolipid synthesis inhibitor, also affects the structures and functions of various organelles, including lysosomes and endoplasmic reticulum (ER). We investigated whether PDMP regulates the mTORC1 activity through its effects on organellar behavior. PDMP induced the translocation of mTORC1 from late endosomes/lysosomes, leading to the dissociation of mTORC1 from its activator Rheb in MC3T3-E1 cells. Surprisingly, we found mTORC1 translocation to the ER upon PDMP treatment. This effect of PDMP was independent of its action as the inhibitor, since two stereoisomers of PDMP, with and without the inhibitor activity, showed essentially the same effect. We confirmed that PDMP inhibits the mTORC1 activity based on the decrease in the phosphorylation of ribosomal S6 kinase, a downstream target of mTORC1, and the increase in LC3 puncta, reflecting autophagosome formation. Furthermore, PDMP inhibited the mTORC1-dependent osteoblastic cell proliferation and differentiation of MC3T3-E1 cells. Accordingly, the present results reveal a novel mechanism of PDMP, which inhibits the mTORC1 activity by inducing the translocation of mTOR from lysosomes to the ER. [ABSTRACT FROM AUTHOR]

Lymphatic vessels (LVs) play critical roles in the maintenance of fluid homeostasis and in pathological conditions, including cancer metastasis. Although mutations in ALK1, a member of the trans- forming growth factor (TGF)-β/bone morphogenetic protein (BMP) receptor family, have been linked to hereditary hemorrhagic tel- angiectasia, a human vascular disease, the roles of activin recep- tor-like kinase 1 (ALK-1) signals in LV formation largely remain to be elucidated. We show that ALK-1 signals inhibit LV formation, and LV5 were enlarged in multiple organs in AIkl-depleted mice. These inhibitory effects of ALK-1 signaling were mediated by BMP- 9, which decreased the number of cultured lymphatic endothetial cells. Bmp9-deficient mouse embryos consistently exhibited en- larged dermal LV5. BMP-9 also inhibited LV formation during inflam- mation and tumorigenesis. BMP-9 downregulated the expression of the transcription factor prospero-related homeobox 1, which is nec- essary to maintain lymphatic endothelial cell identity. Furthermore, silencing prospero-related homeobox 1 expression inhibited lym- phatic endothelial cell proliferation. Our findings reveal a unique molecular basis for the physiological and pathological roles of BMP-9IALK-1 signals in LV formation. [ABSTRACT FROM AUTHOR]

Prostate cancer development is associated with hyperactive androgen signaling. However, the molecular link between androgen receptor (AR) function and humoral factors remains elusive. A prostate cancer mouse model was generated by selectively mutating the AR threonine 877 into alanine in prostatic epithelial cells through Cre-ERT2-mediated targeted somatic mutagenesis. Such AR point mutant mice (ARpe-T877A/Y) developed hypertrophic prostates with responses to both an androgen antagonist and estrogen, although no prostatic tumor was seen. In prostate cancer model transgenic mice, the onset of prostatic tumorigenesis as well as tumor growth was significantly potentiated by introduction of the AR T877A mutation into the prostate. Genetic screening of mice identified Wnt-5a as an activator. Enhanced Wnt-5a expression was detected in the malignant prostate tumors of patients, whereas in benign prostatic hyperplasia such aberrant up-regulation was not obvious. These findings suggest that a noncanonical Wnt signal stimulates development of prostatic tumors with AR hyperfunction. [ABSTRACT FROM AUTHOR]

Chaperone-mediated autophagy (CMA) is a unique proteolytic pathway, in which cytoplasmic proteins recognized by heat shock cognate protein 70 (Hsc70/HSPA8) are transported into lysosomes for degradation. The substrate/chaperone complex binds to the cytosolic tail of the lysosomal-associated membrane protein type 2A (LAMP2A), but whether the interaction between Hsc70 and LAMP2A is direct or mediated by other molecules has remained to be elucidated. The structure of LAMP2A comprises a large lumenal domain composed of two domains, both with the β-prism fold, a transmembrane domain and a short cytoplasmic tail. We previously reported the structural basis for the homophilic interaction of the lumenal domains of LAMP2A, using site-specific photo-crosslinking and/or steric hindrance within cells. In the present study, we introduced a photo-crosslinker into the cytoplasmic tail of LAMP2A and successfully detected its crosslinking with Hsc70, revealing this direct interaction for the first time. Furthermore, we demonstrated that the truncation of the membrane-distal domain within the lumenal domain of LAMP2A reduced the amount of Hsc70 that coimmunoprecipitated with LAMP2A. Our present results suggested that the two-domain architecture of the lumenal domains of LAMP2A underlies the interaction with Hsc70 at the cytoplasmic surface of the lysosome. • The cytoplasmic tail of LAMP2A was directly photo-crosslinked with Hsc70. • Loss of the membrane-distal domain of LAMP2A impaired its interaction with Hsc70. • The assembly of LAMP2A in the lumen may facilitate the recruitment of Hsc70. [ABSTRACT FROM AUTHOR]

Malignant melanoma is one of the untreatable cancers in which conventional therapeutic strategies, including chemotherapy, are hardly effective. Therefore, identification of novel therapeutic targets involved in melanoma progression is urgently needed for developing effective therapeutic methods. Overexpression of interleukin-13 receptor α2 (IL13Rα2) is observed in several cancer types including glioma and pancreatic cancer. Although IL13Rα2 is implicated in the progression of various types of cancer, its expression and roles in the malignant melanoma have not yet been elucidated. In the present study, we showed that IL13Rα2 was expressed in approximately 7.5% melanoma patients. While IL13Rα2 expression in human melanoma cells decreased their proliferation in vitro, it promoted in vivo tumour growth and angiogenesis in melanoma xenograft mouse model. We also found that the expression of amphiregulin, a member of the epidermal growth factor (EGF) family, was correlated with IL13Rα2 expression in cultured melanoma cells, xenograft tumour tissues and melanoma clinical samples. Furthermore, expression of amphiregulin promoted tumour growth, implicating causal relationship between the expression of IL13Rα2 and amphiregulin. These results suggest that IL13Rα2 enhances tumorigenicity by inducing angiogenesis in malignant melanoma, and serves as a potential therapeutic target of malignant melanoma. [ABSTRACT FROM AUTHOR]

Heat shock cognate protein 70 (Hsc70/HSPA8) belongs to the Hsp70 family of molecular chaperones. The fundamental functions of Hsp70 family molecular chaperones depend on ATP-dependent allosteric regulation of binding and release of hydrophobic polypeptide substrates. Hsc70 is also involved in various other cellular functions including selective pathways of protein degradation: chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI), in which Hsc70 recruits substrate proteins containing a KFERQ-like pentapeptide motif from the cytosol to lysosomes and late endosomes, respectively. However, whether the interaction between Hsc70 and the pentapeptide motif is direct or mediated by other molecules has remained unknown. In the present study, we introduced a photo-crosslinker near the KFERQ motif in a CMA/eMI model substrate and successfully detected its crosslinking with Hsc70, revealing the direct interaction between Hsc70 and the KFERQ motif for the first time. In addition, we demonstrated that the loss of the Hsc70 ATPase activity by the D10 N mutation appreciably reduced the crosslinking efficiency. Our present results suggested that the ATP allostery of Hsc70 is involved in the direct interaction of Hsc70 with the KFERQ-like pentapeptide., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Miki Hara-Yokoyama reports financial support was provided by LiberoThera Co., Ltd. Shigeyuki Yokoyama reports a relationship with LiberoThera Co., Ltd that includes: funding grants. Kazue Terasawa reports a relationship with LiberoThera Co., Ltd that includes: employment. There is no patents to disclose relevant to this work. has patent NA pending to NA. There is no additional relationships or activities to declare. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Identification of protein-protein interfaces is necessary for understanding and regulating biological events. Genetic code expansion enables site-specific photo-cross-linking by introducing photo-reactive non-canonical amino acids into proteins at defined positions during translation. This technology is widely used for analyzing protein-protein interactions and is applicable in mammalian cells. However, the identification of the cross-linked region still remains challenging. Our new protocol enables its identification by pre-installing a site-specific cleavage site, an α-hydroxy acid (N ε -allyloxycarbonyl-α-hydroxyl-L-lysine acid, AllocLys-OH), into the target protein. Alkaline treatment cleaves the crosslinked complex at the position of the α-hydroxy acid residue and thus helps to identify which side of the cleavage site, either closer to the N-terminus or C-terminus, the crosslinked site is located on within the target protein. A series of AllocLys-OH introductions narrows down the crosslinked region. This combination of site-specific crosslinking and cleavage promises to be useful for revealing binding interfaces and protein complex geometries. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Search for crosslinkable sites Basic Protocol 2: Site-specific photo-cross-linking/cleavage., (© 2024 Wiley Periodicals LLC.)

Alveologenesis is a spatially coordinated morphogenetic event, during which alveolar myofibroblasts surround the terminal sacs constructed by epithelial cells and endothelial cells (ECs), then contract to form secondary septa to generate alveoli in the lungs. Recent studies have demonstrated the important role of alveolar ECs in this morphogenetic event. However, the mechanisms underlying EC-mediated alveologenesis remain unknown. Herein, we show that ECs regulate alveologenesis by constructing basement membranes (BMs) acting as a scaffold for myofibroblasts to induce septa formation through activating mechanical signaling. Rap1, a small GTPase of the Ras superfamily, is known to stimulate integrin-mediated cell adhesions. EC-specific Rap1-deficient (Rap1 iECKO ) mice exhibit impaired septa formation and hypo-alveolarization due to the decreased mechanical signaling in myofibroblasts. In Rap1 iECKO mice, ECs fail to stimulate integrin β1 to recruit Collagen type IV (Col-4) into BMs required for myofibroblast-mediated septa formation. Consistently, EC-specific integrin β1-deficient mice show hypo-alveolarization, defective mechanical signaling in myofibroblasts, and disorganized BMs. These data demonstrate that alveolar ECs promote integrin β1-mediated Col-4 recruitment in a Rap1-dependent manner, thereby constructing BMs acting as a scaffold for myofibroblasts to induce mechanical signal-mediated alveologenesis. Thus, this study unveils a mechanism of organ morphogenesis mediated by ECs through intrinsic functions., (© 2024. The Author(s).)

The microtubule-associated protein MAP1B has been implicated in axonal growth and brain development. We found that MAP1B is highly expressed in the most aggressive and deadliest breast cancer subtype, triple-negative breast cancer (TNBC), but not in other subtypes. Expression of MAP1B was found to be highly correlated with poor prognosis. Depletion of MAP1B in TNBC cells impairs cell migration and invasion concomitant with a defect in tumorigenesis. We found that MAP1B interacts with key components for invadopodia formation, cortactin, and Tks5, the latter of which is a PtdIns(3,4)P2-binding and scaffold protein that localizes to invadopodia. We also found that Tks5 associates with microtubules and supports the association between MAP1B and α-tubulin. In accordance with their interaction, depletion of MAP1B leads to Tks5 destabilization, leading to its degradation via the autophagic pathway. Collectively, these findings suggest that MAP1B is a convergence point of the cytoskeleton to promote malignancy in TNBC and thereby a potential diagnostic and therapeutic target for TNBC., (© 2024 Inoue et al.)

Tumor progression and metastasis are regulated by endothelial cells undergoing endothelial-mesenchymal transition (EndoMT), a cellular differentiation process in which endothelial cells lose their properties and differentiate into mesenchymal cells. The cells undergoing EndoMT differentiate through a spectrum of intermediate phases, suggesting that some cells remain in a partial EndoMT state and exhibit an endothelial/mesenchymal phenotype. However, detailed analysis of partial EndoMT has been hampered by the lack of specific markers. Transforming growth factor-β (TGF-β) plays a central role in the induction of EndoMT. Here, we showed that inhibition of TGF-β signaling suppressed EndoMT in a human oral cancer cell xenograft mouse model. By using genetic labeling of endothelial cell lineage, we also established a novel EndoMT reporter cell system, the EndoMT reporter endothelial cells (EMRECs), which allow visualization of sequential changes during TGF-β-induced EndoMT. Using EMRECs, we characterized the gene profiles of multiple EndoMT stages and identified CD40 as a novel partial EndoMT-specific marker. CD40 expression was upregulated in the cells undergoing partial EndoMT, but decreased in the full EndoMT cells. Furthermore, single-cell RNA sequencing analysis of human tumors revealed that CD40 expression was enriched in the population of cells expressing both endothelial and mesenchymal cell markers. Moreover, decreased expression of CD40 in EMRECs enhanced TGF-β-induced EndoMT, suggesting that CD40 expressed during partial EndoMT inhibits transition to full EndoMT. The present findings provide a better understanding of the mechanisms underlying TGF-β-induced EndoMT and will facilitate the development of novel therapeutic strategies targeting EndoMT-driven cancer progression and metastasis., (© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

The tumor microenvironment (TME) consists of cancer cells surrounded by stromal components including tumor vessels. Transforming growth factor-β (TGF-β) promotes tumor progression by inducing epithelial-mesenchymal transition (EMT) in cancer cells and stimulating tumor angiogenesis in the tumor stroma. We previously developed an Fc chimeric TGF-β receptor containing both TGF-β type I (TβRI) and type II (TβRII) receptors (TβRI-TβRII-Fc), which trapped all TGF-β isoforms and suppressed tumor growth. However, the precise mechanisms underlying this action have not yet been elucidated. In the present study, we showed that the recombinant TβRI-TβRII-Fc protein effectively suppressed in vitro EMT of oral cancer cells and in vivo tumor growth in a human oral cancer cell xenograft mouse model. Tumor cell proliferation and angiogenesis were suppressed in tumors treated with TβRI-TβRII-Fc. Molecular profiling of human cancer cells and mouse stroma revealed that K-Ras signaling and angiogenesis were suppressed. Administration of TβRI-TβRII-Fc protein decreased the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), interleukin-1β (IL-1β) and epiregulin (EREG) in the TME of oral cancer tumor xenografts. HB-EGF increased proliferation of human oral cancer cells and mouse endothelial cells by activating ERK1/2 phosphorylation. HB-EGF also promoted oral cancer cell-derived tumor formation by enhancing cancer cell proliferation and tumor angiogenesis. In addition, increased expressions of IL-1β and EREG in oral cancer cells significantly enhanced tumor formation. These results suggest that TGF-β signaling in the TME controls cancer cell proliferation and angiogenesis by activating HB-EGF/IL-1β/EREG pathways and that TβRI-TβRII-Fc protein is a promising tool for targeting the TME networks., (© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Genetic code expansion enables site-specific photo-crosslinking by introducing photo-reactive non-canonical amino acids into proteins at defined positions during translation. This technology is widely used for analyzing protein-protein interactions and is applicable in mammalian cells. However, the identification of the crosslinked region still remains challenging. Here, we developed a new method to identify the crosslinked region by pre-installing a site-specific cleavage site, an α-hydroxy acid (N ε -allyloxycarbonyl-α-hydroxyl-l-lysine acid, AllocLys-OH), into the target protein. Alkaline treatment cleaves the crosslinked complex at the position of the α-hydroxy acid residue and thus helps to identify which side of the cleavage site, either closer to the N-terminus or C-terminus, the crosslinked site is located within the target protein. A series of AllocLys-OH introductions narrows down the crosslinked region. By applying this method, we identified the crosslinked regions in lysosomal-associated membrane protein type 2A (LAMP2A), a receptor of chaperone-mediated autophagy, in mammalian cells. The results suggested that at least two interfaces are involved in the homophilic interaction, which requires a trimeric or higher oligomeric assembly of adjacent LAMP2A molecules. Thus, the combination of site-specific crosslinking and site-specific cleavage promises to be useful for revealing binding interfaces and protein complex geometries., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Tumor cells evolve in tumor microenvironment composed of multiple cell types. Among these, endothelial cells (ECs) are the major players in tumor angiogenesis, which is a driver of tumor progression and metastasis. Increasing evidence suggests that ECs also contribute to tumor progression and metastasis as they modify their phenotypes to differentiate into mesenchymal cells through a process known as endothelial-mesenchymal transition (EndoMT). This plasticity of ECs is mediated by various cytokines, including transforming growth factor-β (TGF-β), and modulated by other stimuli depending on the cellular contexts. Recent lines of evidence have shown that EndoMT is involved in various steps of tumor progression, including tumor angiogenesis, intravasation and extravasation of cancer cells, formation of cancer-associated fibroblasts, and cancer therapy resistance. In this review, we summarize current updates on EndoMT, highlight the roles of EndoMT in tumor progression and metastasis, and underline targeting EndoMT as a potential therapeutic strategy., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Transforming growth factor (TGF)-β and its family members, including bone morphogenetic proteins (BMPs), nodal proteins, and activins, are implicated in the development and maintenance of various organs. Here, we review its role in the lymphatic vascular system (the secondary vascular system in vertebrates), which plays a crucial role in various physiological and pathological processes, participating in the maintenance of the normal tissue fluid balance, immune cell trafficking, and fatty acid absorption in the gut. The lymphatic system is associated with pathogenesis in multiple diseases, including lymphedema, inflammatory diseases, and tumor metastasis. Lymphatic vessels are composed of lymphatic endothelial cells, which differentiate from blood vascular endothelial cells (BECs). Although TGF-β family signaling is essential for maintaining blood vessel function, little is known about the role of TGF-β in lymphatic homeostasis. Recently, we reported that endothelial-specific depletion of TGF-β signaling affects lymphatic function. These reports suggest that TGF-β signaling in lymphatic endothelial cells maintains the structure of lymphatic vessels and lymphatic homeostasis, and promotes tumor lymphatic metastasis. Suppression of TGF-β signaling in lymphatic endothelial cells may therefore be effective in inhibiting cancer metastasis. We highlight recent advances in understanding the roles of TGF-β signaling in the formation and maintenance of the lymphatic system., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Itoh and Watabe.)

Transforming growth factor β (TGF-β) increases epithelial cancer cell migration and metastasis by inducing epithelial-mesenchymal transition (EMT). TGF-β also inhibits cell proliferation by inducing G1 phase cell-cycle arrest. However, the correlation between these tumor-promoting and -suppressing effects remains unclear. Here, we show that TGF-β confers higher motility and metastatic ability to oral cancer cells in G1 phase. Mechanistically, keratin-associated protein 2-3 (KRTAP2-3) is a regulator of these dual effects of TGF-β, and its expression is correlated with tumor progression in patients with head and neck cancer and migratory and metastatic potentials of oral cancer cells. Furthermore, single-cell RNA sequencing reveals that TGF-β generates two populations of mesenchymal cancer cells with differential cell-cycle status through two distinctive EMT pathways mediated by Slug/HMGA2 and KRTAP2-3. Thus, TGF-β-induced KRTAP2-3 orchestrates cancer cell proliferation and migration by inducing EMT, suggesting motile cancer cells arrested in G1 phase as a target to suppress metastasis., Competing Interests: Declaration of interests The authors do have a patent related to this work (patent application number [Japan Patent Office]: P2021-214914)., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The blood and lymphatic vasculature networks are not yet fully understood even in mouse because of the inherent limitations of imaging systems and quantification methods. This study aims to evaluate the usefulness of the tissue-clearing technology for visualizing blood and lymphatic vessels in adult mouse. Clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) enables us to capture the high-resolution 3D images of organ- or area-specific vascular structures. To evaluate these 3D structural images, signals are first classified from the original captured images by machine learning at pixel base. Then, these classified target signals are subjected to topological data analysis and non-homogeneous Poisson process model to extract geometric features. Consequently, the structural difference of vasculatures is successfully evaluated in mouse disease models. In conclusion, this study demonstrates the utility of CUBIC for analysis of vascular structures and presents its feasibility as an analysis modality in combination with 3D images and mathematical frameworks., (© 2022. The Author(s).)

Background: During metastasis, cancer cells undergo epithelial-mesenchymal transition (EMT) in response to transforming growth factor-β (TGF-β), which is abundant in the tumor microenvironment, and acquire invasive and metastatic potentials. Metastasis to distant organs requires intravascular invasion and extravasation of cancer cells, which is accompanied by the disruption of the adhesion between vascular endothelial cells. Cancer cell-derived extracellular vesicles (EVs) have been suggested to induce the destabilization of normal blood vessels at the metastatic sites. However, the roles of EVs secreted from cancer cells that have undergone EMT in the destabilization of blood vessels remain to be elucidated. In the present study, we characterized EVs secreted by oral cancer cells undergoing TGF-β-induced EMT and elucidated their effects on the characteristics of vascular endothelial cells., Methods: Induction of EMT by TGF-β in human oral cancer cells was assessed using quantitative RT-PCR (qRT-PCR) and immunocytochemistry. Oral cancer cell-derived EVs were isolated from the conditioned media of oral cancer cells that were treated with or without TGF-β using ultracentrifugation, and characterized using nanoparticle tracking analysis and immunoblotting. The effects of EVs on human umbilical artery endothelial cells were examined by qRT-PCR, cellular staining, and permeability assay. The significant differences between means were determined using a t-test or one-way analysis of variance with Tukey's multiple comparisons test., Results: Oral cancer cells underwent EMT in response to TGF-β as revealed by changes in the expression of epithelial and mesenchymal cell markers at both the RNA and protein levels. Oral cancer cells treated with TGF-β showed increased EV production and altered EV composition when compared with untreated cells. The EVs that originated from cells that underwent EMT by TGF-β induced endothelial-mesenchymal transition, which was characterized by the decreased and increased expression of endothelial and mesenchymal cell markers, respectively. EVs derived from oral cancer cells also induced intercellular gap formation which led to the loss of endothelial cell barrier stability., Conclusions: EVs released from oral cancer cells that underwent TGF-β-induced EMT target endothelial cells to induce vascular destabilization. Detailed characterization of oral cancer-derived EVs and factors responsible for EV-mediated vascular instability will lead to the development of agents targeting metastasis., (© 2022. The Author(s).)

Introduction: Contact hypersensitivity (CHS), a type of delayed-type hypersensitivity, is induced by hapten exposure to the skin and mucosa. We previously reported that, in a murine model of CHS, the vaginal mucosa (VM) sensitization showed lower T-cell responses as compared with the abdominal skin sensitization. To investigate mechanisms of impaired CHS by the VM sensitization, we compared migration of hapten-captured dendritic cells (DCs) in the draining lymph nodes (dLNs) and recruitment of DCs at the sensitized local sites., Methods: Fluorescein isothiocyanate (FITC) or 2,4-dinitrofluorobenzene (DNFB) was used as hapten, and migration of FITC + DCs in the dLNs and local recruitment of MHC class II + and CD11c + cells were compared between abdominal skin and VM sensitization by flow cytometric analyses and immunohistochemistry. Expression of tumor growth factor (TGF)-β at mRNA and protein levels, and local recruitment of CD206 + cells were examined after VM sensitization., Results: VM sensitization showed less numbers of FITC + MHC class II high CD11c + migratory DCs in the dLNs at 6 and 24 h, as compared with skin sensitization. Both skin and VM sensitization induced the recruitment of dermal/submucosal DCs at 6 h, but the number of submucosal DCs in the VM was significantly decreased at 24 h. VM showed persistently higher mRNA levels of TGF-β2/β3 expression than those of the skin before and after sensitization. In the VM sensitization, increment of CD206 + MHC class II + cells was observed especially at the deep lamina propria at 24 h. Most of CD206 + cells were also positive for the binding to Fc chimeric TGF-β receptor that interacts with all TGF-β isoforms, suggesting TGF-β expression., Conclusion: DC migration to dLNs and localization of DCs at the sensitized sites are limited in the VM sensitization. Our results suggest that the existence of TGF-β-expressing CD206 + cells may contribute less sensitization ability and CHS responses in the VM., (© 2022 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

Endothelial-mesenchymal transition (EndoMT), a cellular differentiation process in which endothelial cells (ECs) lose their properties and differentiate into mesenchymal cells, has been observed not only during development but also in various pathological states in adults, including cancer progression and organ/tissue fibrosis. Transforming growth factor-β (TGF-β), an inflammation-related cytokine, has been shown to play central roles in the induction of EndoMT. TGF-β induces EndoMT by regulating the expression of various transcription factors, signaling molecules, and cellular components that confer ECs with mesenchymal characteristics. However, TGF-β by itself is not necessarily sufficient to induce EndoMT to promote the progression of EndoMT-related diseases to a refractory extent. In addition to TGF-β, additional activation by other inflammatory factors is often required to stabilize the progression of EndoMT. Since recent lines of evidence indicate that inflammatory signaling molecules act as enhancers of EndoMT, we summarize the roles of inflammatory factors in the induction of EndoMT and related diseases. We hope that this review will help to develop therapeutic strategies for EndoMT-related diseases by targeting inflammation-mediated EndoMT., (© 2022. The Author(s).)

Cleft palate is one of the major congenital craniofacial birth defects. The etiology underlying the pathogenesis of cleft palate has yet to be fully elucidated. Dissociation of the medial edge epithelium (MEE) at the contacting region of palatal shelves and subsequent migration or apoptosis of MEE cells is required for proper MEE removal. Ras-responsive element-binding protein 1 (RREB1), a RAS transcriptional effector, has recently been shown to play a crucial role in developmental epithelial-mesenchymal transition (EMT), in which loss of epithelial characteristics is an initial step, during mid-gastrulation of embryonic development. Interestingly, the involvement of RREB1 in cleft palate has been indicated in humans. Here, we demonstrated that pan-Ras inhibitor prevents the dissociation of MEE during murine palatal fusion. Rreb1 is expressed in the palatal epithelium during palatal fusion, and knockdown of Rreb1 in palatal organ culture resulted in palatal fusion defects by inhibiting the dissociation of MEE cells. Our present findings provide evidence that RREB1-mediated Ras signaling is required during palatal fusion. Aberrant RREB1-mediated Ras signaling might be involved in the pathogenesis of cleft palate., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Receptor endocytosis is crucial for integrating extracellular stimuli of pro-angiogenic factors, including vascular endothelial growth factor (VEGF), into the cell via signal transduction. VEGF not only triggers various angiogenic events including endothelial cell (EC) migration, but also induces the expression of negative regulators of angiogenesis, including vasohibin-1 (VASH1). While we have previously reported that VASH1 inhibits angiogenesis in vitro and in vivo, its mode of action on EC behavior remains elusive. Recently VASH1 was shown to have tubulin carboxypeptidase (TCP) activity, mediating the post-translational modification of microtubules (MTs) by detyrosination of α-tubulin within cells. However, the role of VASH1 TCP activity in angiogenesis has not yet been clarified. Here, we showed that VASH1 detyrosinated α-tubulin in ECs and suppressed in vitro and in vivo angiogenesis. In cultured ECs, VASH1 impaired endocytosis and trafficking of VEGF receptor 2 (VEGFR2), which resulted in the decreased signal transduction and EC migration. These effects of VASH1 could be restored by tubulin tyrosine ligase (TTL) in ECs, suggesting that detyrosination of α-tubulin negatively regulates angiogenesis. Furthermore, we found that detyrosinated tubulin-rich MTs were not adequate as trafficking rails for VEGFR2 endocytosis. Consistent with these results, inhibition of TCP activity of VASH1 led to the inhibition of VASH1-mediated suppression of VEGF-induced signals, EC migration, and in vivo angiogenesis. Our results indicate a novel mechanism of VASH1-mediated inhibition of pro-angiogenic factor receptor trafficking via modification of MTs.

Lymphangiogenesis, the generation of new lymphatic vessels, plays important roles in cancer metastasis. Outstanding progress during the past decade has dramatically increased the novel knowledge and insights of the mechanisms underlying the generation of new lymphatic vessels, the roles of transcription factors and lymphangiogenic growth factors during physiological development and pathological processes such as cancer and inflammation. Furthermore, an understanding of the molecular consequences during tumor lymphangiogenesis has provided chances to develop better diagnostic and therapeutic approaches that aim to limit the progression of cancer. In this article, we will explain the current knowledge of how lymphatic function is altered in various pathological conditions including cancer progression., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Thyroid transcription factor-1 (TTF-1) is expressed in lung cancer, but its functional roles remain unexplored. TTF-1 gene amplification has been discovered in a part of lung adenocarcinomas, and its action as a lineage-specific oncogene is highlighted. Epithelial-to-mesenchymal transition (EMT) is a crucial event for cancer cells to acquire invasive and metastatic phenotypes and can be elicited by transforming growth factor-beta (TGF-beta). Mesenchymal-to-epithelial transition (MET) is the inverse process of EMT; however, signals that induce MET are largely unknown. Here, we report a novel functional aspect of TTF-1 that inhibits TGF-beta-mediated EMT and restores epithelial phenotype in lung adenocarcinoma cells. This effect was accompanied by down-regulation of TGF-beta target genes, including presumed regulators of EMT, such as Snail and Slug. Moreover, silencing of TTF-1 enhanced TGF-beta-mediated EMT. Thus, TTF-1 can exert a tumor-suppressive effect with abrogation of cellular response to TGF-beta and attenuated invasive capacity. We further revealed that TTF-1 down-regulates TGF-beta2 production in A549 cells and that TGF-beta conversely decreases endogenous TTF-1 expression, suggesting that enhancement of autocrine TGF-beta signaling accelerates the decrease of TTF-1 expression and vice versa. These findings delineate potential links between TTF-1 and TGF-beta signaling in lung cancer progression through regulation of EMT and MET and suggest that modulation of TTF-1 expression can be a novel therapeutic strategy for treatment of lung adenocarcinoma.

Transforming growth factor (TGF)-betas and their family members, including bone morphogenetic proteins (BMPs), Nodal and activins, have been implicated in the development and maintenance of various organs, in which stem cells play important roles. Stem cells are characterized by their ability to self-renew and to generate differentiated cells of a particular tissue, and are classified into embryonic and somatic stem cells. Embryonic stem (ES) cells self-renew indefinitely and contribute to derivatives of all three primary germ layers. In contrast, somatic stem cells, which can be identified in various adult organs, exhibit limited abilities for self-renewal and differentiation in most cases. The multi-lineage differentiation capacity of ES cells and somatic stem cells has opened possibilities for cell replacement therapies for genetic, malignant and degenerative diseases. In order to utilize stem cells for therapeutic applications, it is essential to understand the extrinsic and intrinsic factors regulating self-renewal and differentiation of stem cells. More recently, induced pluripotent stem (iPS) cells have been generated from mouse and human fibroblasts that resemble ES cells via ectopic expression of four transcription factors. iPS cells may have an advantage in regenerative medicine, since they overcome the immunogenicity and ethical controversy of ES cells. Moreover, recent studies have highlighted the involvement of cancer stem cells during the formation and progression of various types of cancers, including leukemia, glioma, and breast cancer. Here, we illustrate the roles of TGF-beta family members in the maintenance and differentiation of ES cells, somatic stem cells, and cancer stem cells.

Lymphangiogenesis is induced by various growth factors, including VEGF-C. Although TGF-beta plays crucial roles in angiogenesis, the roles of TGF-beta signaling in lymphangiogenesis are unknown. We show here that TGF-beta transduced signals in human dermal lymphatic microvascular endothelial cells (HDLECs) and inhibited the proliferation, cord formation, and migration toward VEGF-C of HDLECs. Expression of lymphatic endothelial cell (LEC) markers, including LYVE-1 and Prox1 in HDLECs, as well as early lymph vessel development in mouse embryonic stem cells in the presence of VEGF-A and C, were repressed by TGF-beta but were induced by TGF-beta type I receptor (TbetaR-I) inhibitor. Moreover, inhibition of endogenous TGF-beta signaling by TbetaR-I inhibitor accelerated lymphangiogenesis in a mouse model of chronic peritonitis. Lymphangiogenesis was also induced by TbetaR-I inhibitor in the presence of VEGF-C in pancreatic adenocarcinoma xenograft models inoculated in nude mice. These findings suggest that TGF-beta transduces signals in LECs and plays an important role in the regulation of lymphangiogenesis in vivo.

Vascular endothelial growth factor receptor 2 (VEGFR2) transmits signals of crucial importance to vasculogenesis, including proliferation, migration, and differentiation of vascular progenitor cells. Embryonic stem cell-derived VEGFR2(+) mesodermal cells differentiate into mural lineage in the presence of platelet derived growth factor (PDGF)-BB or serum but into endothelial lineage in response to VEGF-A. We found that inhibition of H-Ras function by a farnesyltransferase inhibitor or a knockdown technique results in selective suppression of VEGF-A-induced endothelial specification. Experiments with ex vivo whole-embryo culture as well as analysis of H-ras(-/-) mice also supported this conclusion. Furthermore, expression of a constitutively active H-Ras[G12V] in VEGFR2(+) progenitor cells resulted in endothelial differentiation through the extracellular signal-related kinase (Erk) pathway. Both VEGF-A and PDGF-BB activated Ras in VEGFR2(+) progenitor cells 5 min after treatment. However, VEGF-A, but not PDGF-BB, activated Ras 6-9 h after treatment, preceding the induction of endothelial markers. VEGF-A thus activates temporally distinct Ras-Erk signaling to direct endothelial specification of VEGFR2(+) vascular progenitor cells.

During embryonic lymphatic development, a homeobox transcription factor Prox1 plays important roles in sprouting and migration of a subpopulation of blood vessel endothelial cells (BECs) toward VEGF-C-expressing cells. However, effects of Prox1 on endothelial cellular behavior remain to be elucidated. Here, we show that Prox1, via induction of integrin alpha9 expression, inhibits sheet formation and stimulates motility of endothelial cells. Prox1-expressing BECs preferentially migrated toward VEGF-C via up-regulation of the expression of integrin alpha9 and VEGF receptor 3 (VEGFR3). In mouse embryos, expression of VEGFR3 and integrin alpha9 is increased in Prox1-expressing lymphatic endothelial cells (LECs) compared with BECs. Knockdown of Prox1 expression in human LECs led to decrease in the expression of integrin alpha9 and VEGFR3, resulting in the decreased chemotaxes toward VEGF-C. These findings suggest that Prox1 plays important roles in conferring and maintaining the characteristics of LECs by modulating multiple signaling cascades and that integrin alpha9 may function as a key regulator of lymphangiogenesis acting downstream of Prox1.

The roles of the transforming growth factor (TGF)-beta superfamily in vasculogenesis have been implicated by the findings that mutations in genes encoding for various TGF-beta superfamily signaling components exhibit defects in vascular tissues in humans and mice. Embryonic stem cell (ESC)-derived vascular progenitor cells have been shown to differentiate into both endothelial and mural cells. We showed that members of the TGF-beta superfamily play important roles during differentiation of vascular progenitor cells derived from mouse ESC. TGF-beta inhibited proliferation and sheet formation of ESC-derived endothelial cells. Interestingly, SB-431542, a synthetic molecule that inhibits the kinases of receptors for TGF-beta and activin, facilitated proliferation and sheet formation of ESC-derived endothelial cells. We also found that stimulation of ESC-derived endothelial cells with TGF-beta resulted in phosphorylation of both Smad2 and Smadl/5; BMP induced phosphorylation of Smad1/5. In this chapter, we present how to study the cellular and biochemical effects of TGF-beta signals on endothelial cells derived from mouse ESCs.

The prostate gland undergoes dramatic changes in growth status during normal physiologic development, following androgen administration to castrate animals, and during tumor development. The prostate stem cell antigen (PSCA, named for its strong sequence homology to the thymocyte marker stem cell antigen 2) is a cell surface molecule associated with human and murine prostate cancer. To help define the regulation of this molecule, we created a transgenic mouse strain, which uses the human PSCA promoter region to control the expression of enhanced green fluorescent protein (GFP). Expression of GFP was detected in mid-gestation following the appearance of prostatic buds from the urogenital sinus. In adult mice, GFP expression was restricted to a subset of cells located in the distal tips of the glands. GFP expression increased during puberty and regeneration driven by androgen and associated with expansive growth of the prostate. GFP-positive cells coexpressed markers associated with both basal and secretory cells in the human prostate. Prostate carcinogenesis driven by T antigen in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model results in an increased percentage and intensity level for PSCA promoter-driven GFP-positive cells. This transgenic system helps define the range of cellular changes associated with altered expression of PSCA, shows that transcriptional control is a major component regulating PSCA levels, and provides a useful tool to study subpopulations of prostate epithelial cells and factors that regulate the PSCA promoter.