Palladin, uusi aktiinitukirangan proteiini

Palladin is a novel actin microfilament associated protein, which together with myotilin and myopalladin forms a novel cytoskeletal IgC2 domain protein family. Whereas the expression of myotilin and myopalladin is limited mainly to striated muscle, palladin is widely expressed in both epithelial and mesenchymal tissues, including heart and the nervous system. Palladin has a complex genetic structure and it is expressed as several different sized and structured splice variants, which also display differences in their expression pattern and interactions. In muscle cells, all the family members localize to the sarcomeric Z-disc, and in non-muscle cells palladin also localizes to the stress-fiber-dense regions, lamellipodia, podosomes and focal adhesions. A common feature of this protein family is the binding to α-actinin, but other interactions are mostly unique to each member. Palladin has been shown to interact with several proteins, including VASP, profilin, Eps8, LASP-1 and LPP. Its domain structure, lack of enzymatic activity and multiple interactions define it as a molecular scaffolding protein, which links together proteins with different functional modalities into large complexes. Palladin has an important role in cytoskeletal regulation, particularly in stress fiber formation and stabilization. This assumption is supported by several experimental results. First, over-expression of palladin in non-muscle cells results in rapid reorganization of the actin cytoskeleton and formation of thick actin bundles. Second, the knock-down of palladin with anti-sense and siRNA techniques or knock-out by genetic methods leads to defective stress fiber formation. Furthermore, palladin is usually up-regulated in situations requiring a highly organized cytoskeleton, such as differentiation of dendritic cells, trophoblasts and myofibroblasts, and activation of astrocytes during glial scar formation. The protein family members have also direct disease linkages; myotilin missense mutations are the cause of LGMD1A and myofibrillar myopathy. Palladin mutations and polymorphisms, on the other hand, have been linked to hereditary pancreatic cancer and myocardial infarction, respectively. In this study we set out to characterize human palladin. We identified several palladin isoforms, studied their tissue distribution and sub-cellular localization. Four novel interaction partners were identified; ezrin, ArgBP2, SPIN90 and Src-kinase.The previously identified interaction between palladin and α-actinin was also characterized in detail. All the identified new binding partners are actin cytoskeleton associated proteins; ezrin links the plasma membrane to the cytoskeleton, ArgBP2 and SPIN90 localize, among other structures, to the lamellipodia and in cardiomyocytes to the Z-disc. Src is a transforming tyrosine kinase, which besides its role in oncogenesis has also important cytoskeletal associations. We also studied palladin in myofibroblasts, which are specialized cells involved in diverse physiological and pathological processes, such as wound healing and tissue fibrosis. We demonstrated that palladin is up-regulated during the differentiation of myofibroblasts in an isoform specific manner, and that this up-regulation is induced by TGF-β via activation of both the SMAD and MAPK signalling cascades. In summary, the results presented here describe the initial characterization of human palladin and offer a basis for further studies.Palladin on hiljattain tunnistettu aktiini tukirangan proteiini, joka yhdessä myotilinin ja myopalladinin kanssa muodostaa uuden proteiiniperheen. Yhteinen piirre perheen jäsenille on rakenteellinen yksikkö, IgC2 domeeni, näitä domeeneja on myotilinissa kaksi, myopalladinissa viisi ja palladinissa kolmesta viiteen. Myotilinia ja myopalladinia ilmennetään pääasiassa poikkijuovaisessa lihaksessa, palladinin ilmentyminen sen sijaan on laajaa käsittäen sekä epiteliaaliset että mesenkymaaliset kudokset, sydänlihaksen ja keskushermoston. Palladinin geneettinen rakenne on monimutkainen ja sitä ilmennetäänkin useana toisistaan sekä kooltaan että rakenteeltaan poikkeavana isoformina. Isoformit eroavat toisistaan lisäksi kudosilmentymisen ja sitoutumiskumppaniensa osalta. Lihassoluissa kaikki proteiiniperheen jäsenet lokalisoituvat sarkomeeriin, tarkemmin ns. Z-juovaan. Muissa solutyypeissä, joissa palladinia ilmennetään se lokalisoituu aktiinista muodostuviin stressisäikeisiin, lamellipodioihin, podosomeihin ja fokaali adheesioihin. Rakenteellisen samankaltaisuuden ohella proteiiniperheen jäsenille on yhteistä myös sitoutuminen tärkeään aktiinitukirangan proteiiniin, α-aktiniiniin, muuten jokaisella jäsenellä on pääasiassa omat sitoutumiskumppaninsa. Palladinilla toistaiseksi tunnettuja kumppaneita ovat mm. ezriini, VASP, profiliini, Eps8, LASP-1 ja LPP. Palladin toimii soluissa todennäköisesti ns. linkkeriproteiinina, joka sitoutumalla samanaikaisesti useaan proteiiniin saattaa nämä yhteen suuremmiksi komplekseiksi. Kyseistä tulkintaa palladinin roolista tukevat mm. sen molekyläärinen rakenne ja entsymaattisen aktiivisuuden puuttuminen. Palladinin solunsisäinen sijoittuminen ja useat koetulokset viittavat siihen että sen pääasiallinen funktio liittyy aktiinitukirangan järjestäytymiseen ja sen ylläpitoon, erityisesti stressisäikeiden osalta. Palladinin kokeellisesti aikaansaatu normaalia runsaampi ilmentyminen soluissa saa aikaan nopean aktiinitukirangan uudelleen järjestäytymisen johon liittyy paksujen aktiinikimppujen muodostuminen. Vastavuoroisesti palladinin ilmentymisen esto ns. anti-sense ja siRNA tekniikoilla johtaa stressisäikeiden hajoamiseen, tämä on osoitettu myös palladinin suhteen poistogeenisen hiiren soluilla tehdyissä kokeissa. Palladinin ilmentyminen lisääntyy tilanteissa, joissa solujen täytyy uudelleen organisoida tukirankansa kuten esimerkiksi trofoblastien, myofibroblastien ja dendriittisten solujen erilaistuessa ja astrosyyttien aktivoituessa glioosissa. Proteiiniperheen jäsenillä on myös suoria tautiyhteyksiä: myotilin geenin mutaatiot aiheuttavat perinnöllisiä lihastauteja: LGMD1A ja MFM ; kun taas tietyn palladinin mutaation on osoitettu liittyvän perinnölliseen haimasyöpään ja polymorfian lisääntyneeseen sydäninfarktin riskiin. Nykyisessä tutkimuksessamme pyrimme selvittämään ihmisen palladinin toimintaa. Tunnistimme useita proteiini isoformeja ja tutkimme niiden ilmentymistä kudoksissa ja soluissa. Kuvasimme neljä uutta sitoutumiskumppania: ezriinin,ArgBP2:n, SPIN90:n ja Src-kinaasin; myös aiemmin tunnistettu sitoutuminen α-aktiniiniin selvitettiin yksityiskohtaisesti. Kaikki uudet sitoutumiskumppanit ovat aktiinitukirankaan liittyviä proteiineja: ezriini liittää solukalvon ja tukirangan yhteen; ArgBP2 ja SPIN90 lokalisoituvat mm. sydänlihassoluissa Z-juovaan kuten palladinkin. Src on tyrosiinikinaasi, jonka hallitsematon aktiivisuus johtaa solujen muuntumiseen pahanlaatuisiksi l. maligniin transformaatioon. Src:lla on myös monenlaisia vaikutuksia aktiini tukirankaan ja se säätelee monien tukiranka proteiinien toimintaa fosforylaation kautta. Tutkimme palladinin toimintaa myös myofibroblasteissa. Nämä fibroblasteista kehittyvät erilaistuneet solut ovat tärkeässä roolissa monissa fysiologisissa ja patologisissa prosesseissa mm. haavan parantumisessa ja kudosfibroosin kehittymisessä. Osoitimme että palladinin määrä lisääntyy erilaistumisprosessin aikana siten että myofibroblastit alkavat ilmentää kookkaampaa palladin isoformia, jota ei tavallisissa fibroblasteissa tavata. Tämän ilmentymisen lisääntymisen saa aikaan kasvutekijä TGF-β, jonka vaikutukset välittyvät SMAD ja MAPK signalointireittien kautta. Soluviljelmillä tehtyjen kokeiden tulokset varmennettiin sekä ihmisen kudosnäytteissä että rotan kokeellisessa haavamallissa. Kuvatut tulokset selvittävät ihmisen palladin proteiinin tehtävää ja muodostavat täten pohjan jatkotutkimuksille

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BAP1 germline variants in Finnish patients with malignant mesothelioma

Objectives: Although asbestos exposure is the most common cause of malignant mesothelioma (MM), an aggressive cancer of the pleura or peritoneum, up to 7% of patients harbor a genetic predisposition to MM. Pathogenic germline variants in the BRCA1-associated protein 1 (BAP1) gene cause a dominantly inherited tumor predisposition syndrome, BAP1-TPDS, in which MM is the second most common associated cancer. Other frequent cancers in BAP1-TPDS are uveal melanoma (UM), cutaneous melanoma and renal cell carcinoma. Additionally patients can exhibit benign skin lesions, BAP1-inactivated nevi (BIN). Most BINs arise sporadically, but patients with BAP1-TPDS may harbor multiple BINs before other tumors or as the only indication of the syndrome. Our objective was to establish the frequency of pathogenic germline BAP1 variants in Finnish patients with MM. Materials and methods: 56 DNA samples archived in the Helsinki Biobank from Finnish patients with MM were sequenced for germline BAP1 variations. Formalin fixed paraffin embedded nevi from a pathogenic variant carrier were subjected to immunohistochemistry and exome sequencing. Results: Sanger sequencing identified one patient with Finnish founder mutation c.1780_1781insT, p. (G549Vfs*49) in BAP1. The carrier was diagnosed with MM over fifteen years before the cohorts mean onset age (mean 68, range 27 to 82) although the patient had no asbestos exposure or family history of BAP1-TPDS. However, the patient had three BINs removed prior to the MM. The c.1780_1781insT is now found from five Finnish BAP1-TPDS families with unknown common ancestor. Conclusion: The frequency of pathogenic germline BAP1 variants in Finnish patients with MM is 1.8 % (95 % CI, 0.04 to 9.2), comparable to the frequency in Finnish patients with UM (1.9 %). The frequency of recurring BINs in patients with BAP1-TPDS should be studied further and genetic testing for BAP1 variants considered if the patient has >= 2 BAP1-TPDS core tumors, including BINs.Peer reviewe

Estrogen receptor beta expression correlates with proliferation in desmoid tumors

Background and objectivesEstrogen receptor signaling and cyclin D1 have a major role in tumor cell proliferation in breast cancer. Desmoid tumors are rare neoplasms that may respond to endocrine treatment. The present study aimed to investigate the expression levels and the clinical relevance of estrogen receptor beta (ER beta) and cyclin D1 in desmoid tumors. MethodsThis study consists of 83 patients with a surgically treated desmoid tumor. ER beta and cyclin D1 expression was examined by immunohistochemistry in tissue microarrays. Cyclin A and Ki67 were studied in our previous work. ResultsMedian ER beta expression was 10.8%. ER beta expression correlated with expression of the proliferation antigens Ki67 (r(p)=0.35, P=0.003), cyclin D1 (r(p)=0.34, P=0.004), and cyclin A (r(p)=0.34, P=0.004). ER beta immunoexpression showed a trend towards predictive impact for recurrence as a continuous variable. Further explorative analysis indicated that very high ER beta expression was related to high risk of relapse (hazard ratio [HR] 2.6; P=0.02).Median cyclin D1 expression was 15.6%. High cyclin D1 expression was associated with high Ki67 and cyclin A expression. Cyclin D1 was not associated with time to recurrence. ConclusionsER beta and cyclin D1 immunopositivity correlated with high proliferation in desmoid tumors. High ER beta expression might be predictive for postoperative recurrence.Peer reviewe

Pirfenidone decreases mesothelioma cell proliferation and migration via inhibition of ERK and AKT and regulates mesothelioma tumor microenvironment in vivo

Malignant mesothelioma is an aggressive cancer with poor prognosis. It is characterized by prominent extracellular matrix, mesenchymal tumor cell phenotypes and chemoresistance. In this study, the ability of pirfenidone to alter mesothelioma cell proliferation and migration as well as mesothelioma tumor microenvironment was evaluated. Pirfenidone is an anti-fibrotic drug used in the treatment of idiopathic pulmonary fibrosis and has also anti-proliferative activities. Mesothelioma cell proliferation was decreased by pirfenidone alone or in combination with cisplatin. Pirfenidone also decreased significantly Transwell migration/ invasion and 3D collagen invasion. This was associated with increased BMP pathway activity, decreased GREM1 expression and downregulation of MAPK/ERK and AKT/mTOR signaling. The canonical Smad-mediated TGF-beta signaling was not affected by pirfenidone. However, pirfenidone blocked TGF-beta induced upregulation of ERK and AKT pathways. Treatment of mice harboring mesothelioma xenografts with pirfenidone alone did not reduce tumor proliferation in vivo. However, pirfenidone modified the tumor microenvironment by reducing the expression of extracellular matrix associated genes. In addition, GREM1 expression was downregulated by pirfenidone in vivo. By reducing two major upregulated pathways in mesothelioma and by targeting tumor cells and the microenvironment pirfenidone may present a novel anti-fibrotic and anti-cancer adjuvant therapy for mesothelioma.Peer reviewe

Gremlin-1 is a key regulator of the invasive cell phenotype in mesothelioma

Peer reviewe

Identification and Validation of Human Papillomavirus Encoded microRNAs

Peer reviewe

Isoform-Specific Regulation of the Actin-Organizing Protein Palladin during TGF-β1-Induced Myofibroblast Differentiation

Contractile myofibroblasts are responsible for remodeling of extracellular matrix during wound healing; however, their continued activity results in various fibrocontractive diseases. Conversion of fibroblasts into myofibroblasts is induced by transforming growth factor-beta1 (TGF-beta1) and is hallmarked by the neo-expression of alpha-smooth muscle actin (alpha-SMA), a commonly used myofibroblast marker. Moreover, myofibroblast differentiation and acquisition of the contractile phenotype involves functionally important alterations in the expression of actin-organizing proteins. We investigated whether myofibroblast differentiation is accompanied by changes in the expression of palladin, a cytoskeletal protein that controls stress fiber integrity. Palladin is expressed as several isoforms, including major 3Ig (90 kDa) and 4Ig (140 kDa) forms that differ in their N-terminal sequence. Expression of the 4Ig isoform is strongly induced in fibroblast stress fibers upon TGF-beta1 treatment preceding alpha-SMA upregulation. TGF-beta1 induced upregulation of palladin is mediated both by Smad and mitogen-activated protein kinase pathways. Furthermore, palladin 4Ig-isoform is co-expressed with alpha-SMA in vivo in experimental rat wounds and in human myofibroblast-containing lesions. Taken together these results identify palladin 4Ig as a novel marker of myofibroblast conversion in vitro and in vivo. They also provide for the first time information about the signaling cascades involved in the regulation of palladin expression

Sytologisille effuusionäytteille uusi tarkempi luokitus

Luokitukset.Nestekertymien sytologisten irtosolututkimusten uuden luokituksen jokaiseen kuuteen ryhmään sisältyvät sytologinen kuvaus, maligniteettiriski ja toimenpidesuositus. Järjestelmä mahdollistaa paremman tiedonkulun patologin ja kliinikon välillä

Sytologisille effuusionäytteille uusi tarkempi luokitus

Nestekertymien sytologisten irtosolututkimusten uuden luokituksen jokaiseen kuuteen ryhmään sisältyvät sytologinen kuvaus, maligniteettiriski ja toimenpidesuositus. Järjestelmä mahdollistaa paremman tiedonkulun patologin ja kliinikon välillä.publishedVersio