以前の論文で、＜mutant IDH1/2の下流にAktのシグナルが動き、さらにポドプラニンの発現が動く＞、という論文があるらしく、その文献の流れにそった形で検証が行われている。我々の論文2報（Mishima_ANP_2006, Motomura_CAS_2012）が引用され、ポドプラニンのWBにおける検出に、NZ-1.2が使用されている。ただ、そのWBのバンドを見ると、本当にポドプラニンのバンドかどうか怪しい。最近、ポドプラニンのバンドというのが、とてもシャープなバンドの論文が多く、しかも、分子量が示されていないので、ポドプラニンのバンドでない可能性がある。今回の論文では、免疫染色ではD2-40を使っているのに、WBではNZ-1.2を使っていることから、D2-40ではバンドが検出されなかったものと考えられる。NZ-1.2の方がD2-40よりも感度が高いのは確かだが、D2-40で検出されなかったため、NZ-1.2をWBのみで使い、出たバンドの一部を切り取った可能性がある。いずれにしても、不可解なデータが多い。Cancerという有名雑誌であるが…。

Mutant IDH1 inhibits PI3K/Akt signaling in human glioma.

Birner P, Pusch S, Christov C, Mihaylova S, Toumangelova-Uzeir K, Natchev S, Schoppmann SF, Tchorbanov A, Streubel B, Tuettenberg J, Guentchev M.

Cancer. 2014 Apr 25. doi: 10.1002/cncr.28732. [Epub ahead of print]　PMID: 24771584 [PubMed - as supplied by publisher]

我々の論文（FEBS2008）が引用されています。

Arch Oral Biol. 2014 Apr 5;59(7):687-694. doi: 10.1016/j.archoralbio.2014.03.017. [Epub ahead of print]

Stromal transforming growth factor-beta 1 is crucial for reinforcing the invasive potential of low invasive cancer.

Hwang YS1, Park KK2, Chung WY3.

Abstract

OBJECTIVE: Tumour cells alter the characteristics of the adjacent stroma to create a supportive microenvironment during cancer progression. In vitro and in vivo experiments were carried out to verify the role of stromal TGF-β1 in reinforcing of the invasive potential in low invasive cancer.

MATERIALS AND METHODS: Isolated NF or CAF was co-cultured with low invasive HSC-2 cells to evaluate whether stromal TGF-β1 induced PDPN expression by Transwell invasion and influenced tumour growth in orthotopic xenografts.

RESULTS: Stimulation by TGF-β1 promoted PDPN expression and Transwell invasion through SMAD signalling as well as activation of Src, P38 mitogen activated protein kinase and extracellular regulated kinase 1/2. PDPN induction was TβRII-dependent. Tumour growth of HSC-2 OSCC in a mouse xenograft was intensified in the tumour CAF microenvironment.

CONCLUSIONS: Stromal TGF-β1 signalling promoted PDPN expression in cancer cells, thereby enhancing tumour growth and leading to a more invasive phenotype.

我々の論文（JBC2007）が引用されています。また、我々が作成したNZ-1.3抗体（e-Bioscience）が使われています。

Human podoplanin-positive monocytes and platelets enhance lymphangiogenesis through the activation of the podoplanin/CLEC-2 axis.(PubMed)

Hur J, Jang JH, Oh IY, Choi JI, Yun JY, Kim J, Choi YE, Ko SB, Kang JA, Kang J, Lee SE, Lee H, Park YB, Kim HS.

Mol Ther. 2014 Apr 16. doi: 10.1038/mt.2014.61. [Epub ahead of print]

Emerging studies suggested that murine podoplanin-positive monocytes (PPMs) are involved in lymphangiogenesis. The goal of this study was to demonstrate the therapeutic lymphangiogenesis of human PPMs by the interaction with platelets. Aggregation culture of human peripheral blood mononuclear cells (PBMCs) resulted in cellular aggregates termed hematospheres. During 5-day culture, PPMs expanded exponentially and expressed several lymphatic endothelial cell-specific markers including VEGF receptor-3 and well-established lymphangiogenic transcription factors. Next, we investigated the potential interaction of PPMs with platelets that had C-type lectin-like receptor-2 (CLEC-2), a receptor of podoplanin. In vitro co-culture of PPMs and platelets stimulated PPMs to strongly express lymphatic endothelial markers and up-regulate lymphangiogenic cytokines. Recombinant human CLEC-2 also stimulated PPMs through Akt and Erk signaling. Likewise, platelets in co-culture with PPMs augmented secretion of a lymphangiogenic cytokine, IL-1beta via podoplanin/CLEC-2 axis. The supernatant obtained from co-culture was able to enhance the migration, viability and proliferation of lymphatic endothelial cell. Local injection of hematospheres with platelets significantly increased lymphatic neovascularization and facilitated wound healing in the full-thickness skin wounds of nude mice. Co-treatment with PPMs and platelets augments lymphangiogenesis through podoplanin/CLEC-2 axis, which thus would be a promising novel strategy of cell therapy to treat human lymphatic vessel disease.Molecular Therapy (2014); doi:10.1038/mt.2014.61.

我々の論文（JI2013）が引用されています。また、我々が作成したNZ-1.2抗体が使われています。

Disulfiram suppresses growth of the malignant pleural mesothelioma cells in part by inducing apoptosis.(PLoS ONE)

Cheriyan VT, Wang Y, Muthu M, Jamal S, Chen D, Yang H, Polin LA, Tarca AL, Pass HI, Dou QP, Sharma S, Wali A, Rishi AK.

PLoS One. 2014 Apr 1;9(4):e93711. doi: 10.1371/journal.pone.0093711. eCollection 2014.PMID: 24690739 [PubMed - in process]

Head Neck. 2014 May 21. doi: 10.1002/hed.23779. [Epub ahead of print]

Prognostic Value of Circulating Tumor Cells with Podoplanin Expression in Patients with Locally Advanced or Metastatic Head and Neck Squamous Cell Carcinoma.

Hsieh JC1, Lin HC, Huang CY, Hsu HL, Wu TM, Lee CL, Chen MC, Wang HM, Tseng CP.

Abstract

Background Podoplanin (PDPN) is a prognostic factor for head and neck squamous cell carcinoma (HNSCC). However, PDPN expression in circulating tumor cells (CTCs) and its prognostic value are not clear. Methods PowerMag system was used to enumerate CTCs from 53 pre-chemotherapy HNSCC patients and 61 healthy donors. PDPN expression was determined by immunofluorescence staining. Results were correlated with clinicopathological parameters and clinical outcome such as patient survival by ROC, univariate and multivariate analyses. Results PDPN was expressed in a subset of CTCs. Both EpCAM+ -CTCs and PDPN+ -CTCs counts were statistically different between disease and non-disease groups (P<0.0001) with no prognostic value. After a median follow-up of 10.5 (6.6-18.5) months, the PDPN+ /EpCAM+ -CTCs ratio>20% was a significant prognostic factor for 6-months death (P=0.011) and was correlated with poor PFS (P=0.016) and OS (P=0.015). Conclusions PDPN+ /EpCAM+ -CTCs ratio is a prognostic factor and defining the ratio in HNSCC patients might be valuable to clinical management.

我々の論文が2つ（ANP2006a,TBI2005）が引用されています。正しい引用です。

Cell Mol Neurobiol. 2014 May 13. [Epub ahead of print]

Up-Regulation of Podoplanin Involves in Neuronal Apoptosis in LPS-Induced Neuroinflammation.

Song Y1, Shen J, Lin Y, Shen J, Wu X, Yan Y, Zhou L, Zhang H, Zhou Y, Cao M, Liu Y.

Abstract

Podoplanin (PDPN) is a mucin-type transmembrane sialoglycoprotein expressed in multiple tissues in adult animals, including the brain, lungs, kidney, and lymphoid organs. Studies of this molecule have demonstrated its great importance in tumor metastasis, platelet aggregation, and lymphatic vessel formation. However, information regarding its regulation and possible function in the central nervous system is still limited. In this study, we performed a neuroinflammatory model by lipopolysaccharide (LPS) lateral ventral injection in adult rats and detected increased expression of PDPN in the brain cortex. Immunofluorescence indicated that PDPN was located in the neurons, but not astrocytes. Moreover, there was a concomitant up-regulation of active caspase-3, cyclin D1, and CDK4 in vivo and vitro studies. In addition, the expression of these three proteins in cortical primary neurons was decreased after knocking down PDPN by siRNA. Collectively, all these results suggested that the up-regulation of PDPN might be involved in neuronal apoptosis in neuroinflammation after LPS injection.

我々の論文が1つ（ANP2006b）が引用されています。オープンアクセスの雑誌です（PLoS ONE）。

PLoS One. 2014 May 5;9(5):e96541. doi: 10.1371/journal.pone.0096541. eCollection 2014.

The role of podoplanin in the biology of differentiated thyroid cancers.

Rudzińska M1, Gaweł D1, Sikorska J1, Karpińska KM1, Kiedrowski M2, Stępień T3, Marchlewska M3, Czarnocka B1.

Abstract

Podoplanin (PDPN), a mucin-type transmembrane glycoprotein specific to the lymphatic system is expressed in a variety of human cancers, and is regarded as a factor promoting tumor progression. The purpose of this study was to elucidate the molecular role of PDPN in the biology of thyroid cancer cells. PDPN expression was evaluated in primary thyroid carcinomas and thyroid carcinoma cell lines by RT-qPCR, Western blotting, IF and IHC. To examine the role of podoplanin in determining a cell's malignant potential (cellular migration, invasion, proliferation, adhesion, motility, apoptosis), a thyroid cancer cell line with silenced PDPN expression was used. We observed that PDPN was solely expressed in the cancer cells of 40% of papillary thyroid carcinoma (PTC) tissues. Moreover, PDPN mRNA and protein were highly expressed in PTC-derived TPC1 and BcPAP cell lines but were not detected in follicular thyroid cancer derived cell lines. PDPN knock-down significantly decreased cellular invasion, and modestly reduced cell migration, while proliferation and adhesion were not affected. Our results demonstrate that PDPN mediates the invasive properties of cells derived from papillary thyroid carcinomas, suggesting that podoplanin might promote PTC progression.

我々の論文が3つ（TBI2005, ANP2006a, ANP2006b）が引用されています。オープンアクセスの雑誌です（Hindawi）。

Biomed Res Int. 2014;2014:132349. doi: 10.1155/2014/132349. Epub 2014 Apr 6.

Expressions of ABCG2, CD133, and Podoplanin in Salivary Adenoid Cystic Carcinoma.

Li W, Tamamura R, Wang B, Liu Q, Liu H, Liu T, Katase N, Xiao J, Nagatsuka H.

Abstract

Adenoid cystic carcinoma (ACC) is one of the most common salivary gland malignant tumors with a high risk of recurrence and metastasis. Current studies on cancer stem cells (CSCs) have verified that CSCs are the driving force behind tumor initiation and progression, suggesting that new cancer therapies may be established by effectively targeting and killing the CSCs. The primary goal of this study is to investigate the expression patterns of ABCG2, CD133, and podoplanin in ACC of minor salivary glands by immunohistochemistry analysis. We found that ABCG2 was weakly expressed in normal looking salivary gland tissues. A significant upregulation of ABCG2 expression in ACC was observed with a similar expression pattern of Ki-67. CD133 was detected in apical membrane of epithelial cells and podoplanin was expressed positively in myoepithelial cells of both normal looking tissue and ACC. However, no significant difference was found of the expression pattern of CD133 and podoplanin between normal looking tissues and ACC. Our observations suggest that CSCs may exist in quiescent cells with ABCG2 positive staining, which are surrounded by cells with positive expression of ABCG2 and Ki-67 in ACC, and costaining with ABCG2 and Ki-67 may help predict the location of CSCs.

D2-40とNZ-1の比較を行った論文です。結論では、＜D2-40のエピトープの失活がおきるが、NZ-1のエピトープについては失活しない＞、と書いてありますが、この結論は間違っています。パラフィン切片上の蛋白質は全体的に失活していきますが、NZ-1のaffinityが高いことによって、多少の蛋白質の分解などが起きても反応するということが正しい見解です。Ogasawara et al., Hybridoma, 2008が引用されています。

Biotech Histochem. 2014 Jun 18:1-7. [Epub ahead of print]

Decreased D2-40 immunoreactivity in stored paraffin sections and methods for preserving it.

Sasaki T1, Kawabata Y, Suzuki N, Tanaka H, Sano M, Kato S, Takemura A, Unno N, Kojima T, Sato K.

Abstract

D2-40, a monoclonal antibody against podoplanin, is a selective marker of lymphatic endothelium and is widely used for research on and diagnosis of pathology of lymphatic vessels. We examined the relation between the duration of tissue section storage and changes in immunostaining by D2-40 antibody; we evaluated also the effects of preservation methods on changes in immunostaining during storage. Staining by D2-40 was attenuated by long-term preservation of scalp skin and lymph node sections at room temperature. The attenuation of D2-40 staining in stored sections was improved by preservation at low temperature, i.e., 4° or - 30° C. We investigated also the immunostaining of preserved tissue sections using NZ-1 and Lyve-1, which are antibodies against lymphatic endothelium markers. Staining by NZ-1 or Lyve-1 antibody was detected clearly in sections that had been stored for 16 weeks. Our study suggests that either long-term storage of D2-40 immunostained tissue sections should be avoided or the section should be preserved at low temperature.

PLoS One. 2014 Jun 16;9(6):e99607. doi: 10.1371/journal.pone.0099607. eCollection 2014.

Clinicopathological Correlations of Podoplanin (gp38) Expression in Rheumatoid Synovium and Its Potential Contribution to Fibroblast Platelet Crosstalk.

Del Rey MJ et al.,

Abstract

INTRODUCTION: Synovial fibroblasts (SF) undergo phenotypic changes in rheumatoid arthritis (RA) that contribute to inflammatory joint destruction. This study was undertaken to evaluate the clinical and functional significance of ectopic podoplanin (gp38) expression by RA SF.

METHODS: Expression of gp38 and its CLEC2 receptor was analyzed by immunohistochemistry in synovial arthroscopic biopsies from RA patients and normal and osteoarthritic controls. Correlation between gp38 expression and RA clinicopathological variables was analyzed. In patients rebiopsied after anti-TNF-α therapy, changes in gp38 expression were determined. Platelet-SF coculture and gp38 silencing in SF were used to analyze the functional contribution of gp38 to SF migratory and invasive properties, and to SF platelet crosstalk.

RESULTS: gp38 was abundantly but variably expressed in RA, and it was undetectable in normal synovial tissues. Among clinicopathologigal RA variables, significantly increased gp38 expression was only found in patients with lymphoid neogenesis (LN), and RF or ACPA autoantibodies. Cultured synovial but not dermal fibroblasts showed strong constitutive gp38 expression that was further induced by TNF-α. In RA patients, anti-TNF-α therapy significantly reduced synovial gp38 expression. In RA synovium, CLEC2 receptor expression was only observed in platelets. gp38 silencing in cultured SF did not modify their migratory and invasive properties but reduced the expression of IL-6 and IL-8 genes induced by SF-platelet interaction.

CONCLUSIONS: In RA, synovial expression of gp38 is strongly associated to LN and it is reduced after anti-TNF-α therapy. Interaction between gp38 and CLEC2 platelet receptor is feasible in RA synovium in vivo and can specifically contribute to gene expression by SF.

我々の論文が英科学誌にアクセプトされました。

Kato Y and Kaneko MK.

A Cancer-specific Monoclonal Antibody Recognizes the Aberrantly Glycosylated Podoplanin.

in press

我々が開発したPMab-1（anti-mouse podoplanin）を使用した論文がPubMedに掲載されました。

The Effect of Podoplanin Inhibition on Lymphangiogenesis under Pathological Conditions.

Maruyama Y, Maruyama K, Kato Y, Kajiya K, Moritoh S, Yamamoto K, Matsumoto Y, Sawane M, Kerjaschki D, Nakazawa T, Kinoshita S.

Invest Ophthalmol Vis Sci. 2014 Jul 1. pii: IOVS-13-13711. doi: 10.1167/iovs.13-13711. [Epub ahead of print]

PMID: 24985477

Purpose: Podoplanin has been shown to be a reliable marker of lymphatic endothelium, but its role in the lymphatic system has not been well investigated. The purpose of this study is to investigate the role of podoplanin in lymphangiogenesis and macrophage functions under inflammatory conditions. Methods: Mouse corneal suture and ear section models were used to induce lymphangiogenesis and macrophage infiltration. Anti-LYVE-1 antibody was used to visualize lymphatic vessels. Thioglycollate-induced macrophages (mps) were collected and cultured with LPS, IFNγ, and anti-mouse podoplanin antibody (PMab-1). Podoplanin, NF- κB, and MAP kinase pathway expression were detected by Western blot analysis. TNFα secretion was measured by ELISA. Results: Administration of PMab-1, reduced lymphangiogenesis both in the corneal suture and ear wound healing models. Also, PMab-1 suppressed mps infiltration at the site of wound healing. Moreover, administration of PMab-1 led to a significant suppression of the rejection reaction in the corneal transplantation model. Our in vitro experiments showed that PMab-1 suppressed TNFα secretion from mps under inflamed conditions, especially secretion caused by LPS stimulation. We confirmed the effect of PMab-1 on mps under inflamed conditions with a Western blot experiment, which clearly showed that the phosphorylation signal of both the MAPK and NF-kB pathways was suppressed by PMab-1. Conclusions: Podoplanin neutralization resulted in inhibition of lymphatic growth associated with corneal and ear wound healing as well as mps inflammation. These data suggest that podoplanin is a novel therapeutic target for suppressing lymphangiogenesis and inflammation.

我々が開発したPMab-1（anti-mouse podoplanin）を使用した論文がInvest Ophthalmol Vis Sci.に掲載されました。眼科との共同研究です。ポドプラニンの命名者であるDr. Kerjaschki も共著になっています。

Maruyama Y, Maruyama K, Kato Y, Kajiya K, Moritoh S, Yamamoto K, Matsumoto Y, Kerjaschki D, Nakazawa T, Kinoshita S.

The Effect of Podoplanin Inhibition on Lymphangiogenesis under Pathological Conditions.

Invest Ophthalmol Vis Sci., in press

我々の論文が10報引用されています。 Med Oncol. 2014 Sep;31(9):24. doi: 10.1007/s12032-014-0024-6. Epub 2014 Aug 21.

Podoplanin: a novel regulator of tumor invasion and metastasis.

Dang Q, Liu J, Li J, Sun Y.

Abstract

Podoplanin, a small mucin-type sialoglycoprotein, was recently shown to be involved in tumor progression. Podoplanin is overexpressed in cancer cells of various human malignancies, and recently, it is also detected in intratumoral stromal cells. We now appreciate that podoplanin plays a dual role in cancer: it can not only suppress tumor growth but also promote tumor progression. Researchers have identified several potential pathways invoked by podoplanin, which participate in the epithelial-to-mesenchymal transition, collective-cell migration, platelet activation and aggregation, and lymphangiogenesis, and thus regulate the tumor invasion and metastasis. Here, we discuss the current experimental and human clinical data on podoplanin to validate the multiple context-dependent functions in different microenvironments and to delineate the diverse regulatory mechanisms.

我々の論文が2報引用されています。

Cancer Med. 2014 Aug 16. doi: 10.1002/cam4.320. [Epub ahead of print]

Suppression of Aggrus/podoplanin-induced platelet aggregation and pulmonary metastasis by a single-chain antibody variable region fragment.

Miyata K, Takagi S, Sato S, Morioka H, Shiba K, Minamisawa T, Takami M, Fujita N.

Abstract

Almost all highly metastatic tumor cells possess high platelet aggregating abilities, thereby form large tumor cell-platelet aggregates in the microvasculature. Embolization of tumor cells in the microvasculature is considered to be the first step in metastasis to distant organs. We previously identified the platelet aggregation-inducing factor expressed on the surfaces of highly metastatic tumor cells and named as Aggrus. Aggrus was observed to be identical to the marker protein podoplanin (alternative names, T1α, OTS-8, and others). Aggrus is frequently overexpressed in several types of tumors and enhances platelet aggregation by interacting with the platelet receptor C-type lectin-like receptor 2 (CLEC-2). Here, we generated a novel single-chain antibody variable region fragment (scFv) by linking the variable regions of heavy and light chains of the neutralizing anti-human Aggrus monoclonal antibody MS-1 with a flexible peptide linker. Unfortunately, the generated KM10 scFv failed to suppress Aggrus-induced platelet aggregation in vitro. Therefore, we performed phage display screening and finally obtained a high-affinity scFv, K-11. K-11 scFv was able to suppress Aggrus-induced platelet aggregation in vitro. Moreover, K-11 scFv prevented the formation of pulmonary metastasis in vivo. These results suggest that K-11 scFv may be useful as metastasis inhibitory scFv and is expected to aid in the development of preclinical and clinical examinations of Aggrus-targeted cancer therapies.

Bloodの常連のWatsonラボからの論文です。

Blood. 2014 Oct 28. pii: blood-2014-05-579375. [Epub ahead of print]

The N-terminal SH2 domain of Syk is required for (hem)ITAM but not integrin signalling in mouse platelets.

Hughes CE, Finney BA, Koentgen F, Lowe KL, Watson SP.

Abstract

We have used a novel knock-in mouse to investigate the effect of disruption of phospho-tyrosine binding of the N-terminal SH2 domain of Syk on platelet activation by GPVI, CLEC-2 and integrin αIIbβ3. The SykR41Afl/fl mouse was crossed to a PF4-Cre+ mouse to induce expression of the Syk mutant in the megakaryocyte/platelet lineage. SykR41Afl/fl;PF4-Cre mice are born at approximately 50% of the expected frequency and have a similar phenotype to Sykfl/fl;PF4-Cre mice, including blood-lymphatic mixing and chyloascites. Anastomosis of the venous and lymphatic vasculatures can be seen in the mesenteric circulation accounting for rapid and continuous mixing of the two vasculatures. Platelet activation by CLEC-2 and GPVI is abolished in SykR41Afl/fl;PF4-Cre platelets. Syk phosphorylation on Tyr519/20 is blocked in CLEC-2 stimulated platelets suggesting a model in which binding of Syk via its N-terminal SH2 domain regulates autophosphorylation. In contrast, outside-in signalling by integrin αIIbβ3 is not altered but is inhibited in the presence of inhibitors of Src and Syk tyrosine kinases. These results demonstrate that in mice, αIIbβ3 regulates Syk through an ITAM-independent pathway, and provide novel insight into the course of events underlying Syk activation and hemITAM phosphorylation by CLEC-2.

同じグループからの論文が Nat Immunol.に出ました。我々の論文が１報引用されています。

Nat Immunol. 2014 Oct 27. doi: 10.1038/ni.3035. [Epub ahead of print]

The CLEC-2-podoplanin axis controls the contractility of fibroblastic reticular cells and lymph node microarchitecture.

Acton SE, Farrugia AJ, Astarita JL, Mourão-Sá D, Jenkins RP, Nye E, Hooper S, van Blijswijk J, Rogers NC, Snelgrove KJ, Rosewell I, Moita LF, Stamp G, Turley SJ, Sahai E, Reis e Sousa C.

Abstract

In lymph nodes, fibroblastic reticular cells (FRCs) form a collagen-based reticular network that supports migratory dendritic cells (DCs) and T cells and transports lymph. A hallmark of FRCs is their propensity to contract collagen, yet this function is poorly understood. Here we demonstrate that podoplanin (PDPN) regulates actomyosin contractility in FRCs. Under resting conditions, when FRCs are unlikely to encounter mature DCs expressing the PDPN receptor CLEC-2, PDPN endowed FRCs with contractile function and exerted tension within the reticulum. Upon inflammation, CLEC-2 on mature DCs potently attenuated PDPN-mediated contractility, which resulted in FRC relaxation and reduced tissue stiffness. Disrupting PDPN function altered the homeostasis and spacing of FRCs and T cells, which resulted in an expanded reticular network and enhanced immunity.

またしても、ポドプラニン関連のすごい論文が Natureに出ました。

Nature. 2014 Oct 22;514(7523):498-502. doi: 10.1038/nature13814.

Dendritic cells control fibroblastic reticular network tension and lymph node expansion.

Astarita JL, Cremasco V, Fu J, Darnell MC, Peck JR, Nieves-Bonilla JM, Song K, Kondo Y, Woodruff MC, Gogineni A, Onder L, Ludewig B, Weimer RM, Carroll MC, Mooney DJ, Xia L, Turley SJ.

Abstract

After immunogenic challenge, infiltrating and dividing lymphocytes markedly increase lymph node cellularity, leading to organ expansion. Here we report that the physical elasticity of lymph nodes is maintained in part by podoplanin (PDPN) signalling in stromal fibroblastic reticular cells (FRCs) and its modulation by CLEC-2 expressed on dendritic cells. We show in mouse cells that PDPN induces actomyosin contractility in FRCs via activation of RhoA/C and downstream Rho-associated protein kinase (ROCK). Engagement by CLEC-2 causes PDPN clustering and rapidly uncouples PDPN from RhoA/C activation, relaxing the actomyosin cytoskeleton and permitting FRC stretching. Notably, administration of CLEC-2 protein to immunized mice augments lymph node expansion. In contrast, lymph node expansion is significantly constrained in mice selectively lacking CLEC-2 expression in dendritic cells. Thus, the same dendritic cells that initiate immunity by presenting antigens to T lymphocytes also initiate remodelling of lymph nodes by delivering CLEC-2 to FRCs. CLEC-2 modulation of PDPN signalling permits FRC network stretching and allows for the rapid lymph node expansion--driven by lymphocyte influx and proliferation--that is the critical hallmark of adaptive immunity.

J Clin Invest. 2014 Nov 21. pii: 74685. doi: 10.1172/JCI74685. [Epub ahead of print]（PubMed）

Podoplanin negatively regulates CD4+ effector T cell responses.

Peters A, Burkett PR, Sobel RA, Buckley CD, Watson SP, Bettelli E, Kuchroo VK.

Abstract

Podoplanin (PDPN, also known as Gp38) is highly expressed on the surface of lymphatic endothelial cells, where it regulates development of lymphatic vessels. We have recently observed that PDPN is also expressed on effector T cells that infiltrate target tissues during autoimmune inflammation; however, the function of PDPN in T cells is largely unclear. Here, we demonstrated that global deletion of Pdpn results in exaggerated T cell responses and spontaneous experimental autoimmune encephalomyelitis (EAE) in mice with a susceptible genetic background. In contrast, T cell-specific overexpression of PDPN resulted in profound defects in IL-7-mediated T cell expansion and survival. Consequently, these animals exhibited a more rapid resolution of CNS inflammation, characterized by a reduced effector CD4+ T cell population in the CNS. Mice harboring a T cell-specific deletion of Pdpn developed exacerbated EAE, with increased accumulation of effector CD4+ T cells in the CNS. Transcriptional profiling of naturally occurring PDPN+ effector T cells in the CNS revealed increased expression of other inhibitory receptors, such as Pd1 and Tim3, and decreased expression of prosurvival factors, including Il7ra. Together, our data suggest that PDPN functions as an inhibitory molecule on T cells, thereby promoting tissue tolerance by limiting long-term survival and maintenance of CD4+ effector T cells in target organs.

Clin Cancer Res. 2014 Nov 11. pii: clincanres.0846.2014. [Epub ahead of print]（PubMed）

Podoplanin-positive cancer-associated fibroblasts in the tumor microenvironment induce primary resistance to EGFR-TKIs in lung adenocarcinoma with EGFR mutation.

Yoshida T, Ishii G, Goto K, Neri S, Hashimoto H, Yoh K, Niho S, Umemura S, Matsumoto S, Ohmatsu H, Iida S, Niimi A, Nagai K, Ohe Y, Ochiai A.

Abstract

Purpose: The biological characteristics of microenvironmental constituents, especially cancer-associated fibroblasts (CAFs), can be key regulators of the cellular sensitivity to molecular-targeted therapy. EGFR tyrosine kinase inhibitors (EGFR-TKIs) have marked therapeutic effects against non-small cell lung cancer (NSCLC) with EGFR mutations, but some patients have exhibited primary resistance to EGFR-TKIs. We recently reported that podoplanin (PDPN)-positive fibroblasts are associated with a tumor-promoting phenotype of CAFs in lung adenocarcinoma. The aim of this study was to evaluate whether the susceptibility of NSCLC to EGFR-TKIs could be affected by PDPN-expressing CAFs. Experimental Design: We evaluated the EGFR-TKI sensitivity of EGFR-mutant lung adenocarcinoma cell lines co-cultured with PDPN-expressing CAFs. We also examined the association between the expression of PDPN in CAFs in surgical specimens and EGFR-TKI response of postoperative recurrent patients with EGFR mutations (N = 106). Results: Lung adenocarcinoma cell lines became more resistant to EGFR-TKI when co-cultured with PDPN-expressing CAFs, compared with control CAFs in vitro. The knockdown of PDPN-expression on CAFs cancelled the resistance to EGFR-TKIs in cancer cells. Compared with control CAFs, the cancer cells that were co-cultured with PDPN-positive CAFs continued to exhibit significantly higher p-ERK levels after treatment with gefitinib. Furthermore, postoperative recurrent patients with PDPN-positive CAFs had a significantly lower overall response rate to EGFR-TKIs, compared with those with PDPN-negative CAFs (53% vs 83%, P < 0.01). Conclusions: PDPN-positive CAFs plays an important role in primary resistance to EGFR-TKIs and may be an ideal therapeutic target for use in combination therapy with EGFR-TKIs.

理研との共同研究がStructureに掲載されました。

Nagae M, Morita-Matsumoto K, Kato M, Kaneko MK, Kato Y, Yamagichi Y.

A novel platform of C-type lectin-like receptor CLEC-2 for binding O-glycosylated podoplanin and non-glycosylated rhodocytin.

Structure, on line, DOI: http://dx.doi.org/10.1016/j.str.2014.09.009

再び、Watsonラボからの論文です。彼らは私達の論文をいつもは引用しないのですが、今回は３つも引用しています。また、eBioscienceから販売してもらっているNZ-1.3（我々の開発したNZ-1シリーズ）も使っています。タイトルや要旨を読んでも、他の彼らの論文との区別が全くできない程似ているのですが、このように常に論文を出し続ける姿勢は見習わなくてはなりません。彼らはCLEC-2の発見者ですが、CLEC2とポドプラニンとの結合の発見は、山梨大学と我々の共同研究の成果です。しかし、論文を出し続けると、その人達の成果と勘違いされることも多いのは事実です。我々も頑張らなくてはなりません。

J Biol Chem. 2014 Nov 3. pii: jbc.M114.584284. [Epub ahead of print]

Syk and Src family kinases regulate CLEC-2 mediated clustering of Podoplanin and platelet adhesion to lymphatic endothelial cells.

Pollitt AY, Poulter NS, Gitz E, Navarro-Nu Ntildeez L, Wang YJ, Hughes CE, Thomas SG, Nieswandt B, Douglas MR, Owen DM, Jackson DG, Dustin ML, Watson SP.

Abstract

The interaction of CLEC-2 on platelets with Podoplanin on lymphatic endothelial cells initiates platelet signalling events that are necessary for prevention of blood-lymph mixing during development. In the present study, we show that CLEC-2 signalling via Src family and Syk tyrosine kinases promotes platelet adhesion to primary mouse lymphatic endothelial cells at low shear. Using supported lipid bilayers containing mobile Podoplanin, we further show that activation of Src and Syk in platelets promotes clustering of CLEC-2 and Podoplanin. Clusters of CLEC-2-bound Podoplanin migrate rapidly to the centre of the platelet to form a single structure. Fluorescence life-time imaging demonstrates that molecules within these clusters are within 10 nm of one another and that the clusters are disrupted by inhibition of Src and Syk family kinases. CLEC-2 clusters are also seen in platelets adhered to immobilised Podoplanin using direct stochastic optical reconstruction microscopy (dSTORM). These findings provide mechanistic insight by which CLEC-2 signalling promotes adhesion to Podoplanin and regulation of Podoplanin signalling thereby contributing to lymphatic vasculature development.

LpMab-3の解析論文（Oki et al）がMonoclon. Antib. Immunodiagn. Immunother.にアクセプトされました。

理研との共同研究の論文が、PubMedに掲載されました。
A Platform of C-Type Lectin-like Receptor CLEC-2 for Binding O-Glycosylated Podoplanin and Nonglycosylated Rhodocytin.