Role of the canonical transient receptor potential channel 2 (TRPC2) in thyroid cell function

Kalciumjonen reglerar flera processer i celler såsom transkribering av gener, celldelning, cellernas rörlighet och celldöd. Därför har cellerna utvecklat många mekanismer för att reglera den intracellulära kalciumkoncentrationen. Kalciumkanaler spelar en viktig roll i denna regleringsprocess. TRPC-kanalerna (eng. canonical transient receptor potential) är en familj av jonkanaler med sju medlemmar (TRPC1-7) vars regleringsmekanismer och fysiologiska roller är varierande. TRPC2-kanalens fysiologiska signifikans, samt hur kanalen regleras, är dåligt karakteriserad. För första gången, rapporterar vi närvaron av TRPC2 kanalen i råttans sköldkörtelceller samt primära sköldkörtelceller från råtta. Hos gnagare har TRPC2 antagits vara exklusivt uttryckt i det vomeronasala organet. För att undersöka den fysiologiska betydelsen av kanalen, har vi utvecklat stabila celler med nedreglerat TRPC2 (shTRPC2) m.h.a. shRNA-teknik. Nedreglering av TRPC2 resulterade i stora skillnader i flera viktiga cellulära funktioner och i regleringen av sköldkörtelcellernas cellsignalering. Nedreglering av TRPC2 orsakade minskad agonist-beroende frigivning av kalcium från det endoplasmatiska nätverket, samt minskat agonist-beroende inflöde av extracellulärt kalcium, men ökade det basala kalciuminflödet. Uttrycket av PKCβ1 och PKCδ, SERCA-aktiviteten och kalciumhalten i det endoplasmatiska nätverket minskade i shTRPC2 celler. Kommunikation mellan kalcium- och cAMP-signalering påvisades vara TRPC2-beroende, vilket visades reglera uttrycket av TSH-receptorn. Vi undersökte också betydelsen av TRPC2 kanalen i reglering av sköldkörtelcellers proliferation, migration, vidhäftning och invasion; processer som alla var dämpade i shTRPC2 celler. Samamnfattningsvis påvisade dessa resultat en ny och viktig fysiologisk betydelse för TRPC2 kanalerna.

On Glucose Metabolism in Patients with the m.3243A>G Mutation

Background: The m.3243A>G mutation in mitochondrial DNA is the most common cause for mitochondrial diabetes. In addition, unexpected deaths related to the m.3243A>G associate with encephalopathy and cardiomyopathy. Failing mitochondrial respiratory chain in neurons, myocytes and beta cells is considered to underlie the multiorgan manifestations of the m.3243A>G. Aims: The primary aim of the study was to characterize the organ-specific glucose metabolism in patients with m.3243A>G and secondly, to study patients with or without signs of diabetes, cardiomyopathy or encephalopathy. The insulin-stimulated glucose metabolism in brain, heart, skeletal muscle, adipose tissue and liver were measured with 2-deoxy-2-[18F]fluoro-α-D-glucose in 15 patients and 14 controls. Brain oxygen metabolism was assessed with [15O]oxygen and insulin secretion was modelled based on oral glucose tolerance test. Results: The glucose oxidation in brain was globally decreased in patients with or without clinical encephalopathy. The insulin-stimulated glucose influx to skeletal muscle and adipose tissue was decreased in patients with or without diabetes as the hepatic glucose metabolism was normal. Impaired beta cell function and myocardial glucose uptake were associated with the high m.3243A>G heteroplasmy. Conclusions: This cross-sectional study suggests that: 1) The ability of insulin to stimulate glucose metabolism in skeletal muscle and adipose tissue is weakened before the beta cell failure results in mitochondrial diabetes. 2) Glucose oxidation defect is detected in otherwise unaffected cerebral regions in patients with the m.3243A>G, thus it likely precedes the clinical encephalopathy. 3) Uneconomical glucose hypometabolism during hyperinsulinemia contributes to the cardiac vulnerability in patients with high m.3243A>G heteroplasmy

Virus Infections in Early Childhood, Cow’s Milk Formula Exposure and Genetic Predisposition in the Development of Diabetes-Associated Autoimmunity

Varhaislapsuuden virusinfektioiden, lehmänmaitopohjaisen äidinmaitovastikeen ja geneettisen alttiuden merkitys diabetekseen liittyvän autoimmuniteetin kehittymisessä Tyypin 1 diabetes on autoimmuunisairaus, joka syntyy haiman insuliinia tuottavien beta-solujen tuhouduttua elimistön oman immuunipuolustusjärjestelmän hyökkäyksen seurauksena. Sekä perimän että ympäristötekijöiden arvellaan vaikuttavan tautiprosessiin, mutta taudin tarkkaa syntymekanismia ei tunneta. Tutkimuksen tarkoituksena oli selvittää varhaislapsuuden ympäristötekijöiden vaikutusta beta-soluautoimmuniteetin syntyyn, erityispaino tutkimuksessa oli ympäristötekijöiden yhteisvaikutuksessa sekä geneettisten riskitekijöiden ja ympäristötekijöiden vuorovaikutuksessa. Varhaislapsuudessa sairastettu sytomegalovirus- tai enterovirusinfektio ei lisännyt beta-soluautoimmuniteetin riskiä lapsilla, joilla on geneettisesti kohonnut riski sairastua tyypin 1 diabetekseen. Ennen puolen vuoden ikää sairastettu rotavirusinfektio lisäsi hieman tyypin 1 diabetekseen liittyvän autoimmuniteetin riskiä. Tarkemmassa analyysissa varhaislapsuuden enterovirusinfektio osoittautui kuitenkin autovasta-aineiden muodostumisen riskitekijäksi niiden lasten joukossa, jotka olivat saaneet lehmänmaitopohjaista äidinmaidon vastiketta ensimmäisten elinkuukausien aikana. Tämä löydös viittaa enterovirusinfektion ja lehmänmaitopohjaisen vastikkeen yhteisvaikutukseen tyypin 1 diabetekseen liittyvän autoimmuniteetin synnyssä. Löydösten mukaan PTPN22 geenin C1858T polymorfismi vaikuttaa CD4+ T solujen aktivaatioon ja proliferaatiovasteeseen, 1858T alleeliin liittyy alentunut T-soluresepto-rivälitteinen aktivaatio. 1858T alleelin kantajuuteen liittyy lisäksi lisääntynyt autovasta-aineiden ja kliinisen diabeteksen ilmaantuvuus. Tämä yhteys rajoittui yksilöihin, jotka olivat altistuneet lehmänmaitopohjaiselle vastikkeelle ennen kuuden kuukauden ikää. Tulosten mukaan sekä ympäristötekijöiden väliset yhteisvaikutukset että perimä vaikuttavat yksittäisen ympäristötekijän merkitykseen tyypin 1 diabetekseen liittyvän autoimmuniteetin synnyssä. Nämä yhteisvaikutukset ympäristötekijöiden kesken ja perimän ja ympäristötekijöiden välillä selittävät aiemmin julkaistujen tulosten ristiriittaisuutta tutkimuksissa, joissa on analysoitu vain yhden ympäristötekijän vaikutusta diabeteksen ilmaantuvuuteen.

Probing MST1 Kinase Sequence and Structure for Rational Drug Discovery (Targeting diabetes by blocking protein-protein interactions)

Apoptotic beta cell death is an underlying cause majorly for type I and to a lesser extent for type II diabetes. Recently, MST1 kinase was identified as a key apoptotic agent in diabetic condition. In this study, I have examined MST1 and closely related kinases namely, MST2, MST3 and MST4, aiming to tackle diabetes by exploring ways to selectively block MST1 kinase activity. The first investigation was directed towards evaluating possibilities of selectively blocking the ATP binding site of MST1 kinase that is essential for the activity of the enzymes. Structure and sequence analyses of this site however revealed a near absolute conservation between the MSTs and very few changes with other kinases. The observed residue variations also displayed similar physicochemical properties making it hard for selective inhibition of the enzyme. Second, possibilities for allosteric inhibition of the enzyme were evaluated. Analysis of the recognized allosteric site also posed the same problem as the MSTs shared almost all of the same residues. The third analysis was made on the SARAH domain, which is required for the dimerization and activation of MST1 and MST2 kinases. MST3 and MST4 lack this domain, hence selectivity against these two kinases can be achieved. Other proteins with SARAH domains such as the RASSF proteins were also examined. Their interaction with the MST1 SARAH domain were evaluated to mimic their binding pattern and design a peptide inhibitor that interferes with MST1 SARAH dimerization. In molecular simulations the RASSF5 SARAH domain was shown to strongly interact with the MST1 SARAH domain and possibly preventing MST1 SARAH dimerization. Based on this, the peptidic inhibitor was suggested to be based on the sequence of RASSF5 SARAH domain. Since the MST2 kinase also interacts with RASSF5 SARAH domain, absolute selectivity might not be achieved.

Regulation of Spermatogenesis: Differentiation of GFP-labeled Stem Cells and the Function of Cytoplasmic Bridges

During spermatogenesis, different genes are expressed in a strictly coordinated fashion providing an excellent model to study cell differentiation. Recent identification of testis specific genes and the development of green fluorescence protein (GFP) transgene technology and an in vivo system for studying the differentiation of transplanted male germ cells in infertile testis has opened new possibilities for studying the male germ cell differentiation at molecular level. We have employed these techniques in combination with transillumination based stage recognition (Parvinen and Vanha-Perttula, 1972) and squash preparation techniques (Parvinen and Hecht, 1981) to study the regulation of male germ cell differentiation. By using transgenic mice expressing enhanced-(E)GFP as a marker we have studied the expression and hormonal regulation of beta-actin and acrosin proteins in the developmentally different living male germ cells. Beta-actin was demonstrated in all male germ cells, whereas acrosin was expressed only in late meiotic and in postmeiotic cells. Follicle stimulating hormone stimulated b-actin-EGFP expression at stages I-VI and enhanced the formation of microtubules in spermatids and this way reduced the size of the acrosomic system. When EGFP expressing spermatogonial stem cells were transplanted into infertile mouse testis differentiation and the synchronized development of male germ cells could be observed during six months observation time. Each colony developed independently and maintained typical stage-dependent cell associations. Furthermore, if more than two colonies were fused, each of them was adjusted to one stage and synchronized. By studying living spermatids we were able to demonstrate novel functions for Golgi complex and chromatoid body in material sharing between neighbor spermatids. Immunosytochemical analyses revealed a transport of haploid cell specific proteins in spermatids (TRA54 and Shippo1) and through the intercellular bridges (TRA54). Cytoskeleton inhibitor (nocodazole) demonstrated the importance of microtubules in material sharing between spermatids and in preserving the integrity of the chromatoid body. Golgi complex inhibitor, brefeldin A, revealed the great importance of Golgi complex i) in acrosomic system formation ii) TRA54 translation and in iii) granule trafficking between spermatids.

Role and Function of c-Jun Protein Complex in Cancer Cell Behavior

Transcription factors play a crucial role in the regulation of cell behavior by modulating gene expression profiles. Previous studies have described a dual role for the AP-1 family transcription factor c-Jun in the regulation of cellular fate. In various cell types weak and transient activations of c-Jun N-terminal kinase (JNK) and c-Jun appear to contribute to proliferation and survival, whereas strong and prolonged activation of JNK and c-Jun result in apoptosis. These opposite roles played by c-Jun are cell type specific and the molecular mechanisms defining these antonymous c-Jun-mediated responses remain incompletely understood. c-Jun activity in transformed cells is regulated by signalling cascades downstream of oncoproteins such as Ras and Raf. In addition, the pro-proliferative role and the survival promoting function for c-Jun has been described in various cancer models. Furthermore, c-Jun was described to be overexpressed in different cancer types. However, the molecular mechanisms by which c-Jun exerts these oncogenic functions are not all clearly established. Therefore it is of primary interest to further identify molecular mechanisms and functions for c-Jun in cancer. Regulation of gene expression is tightly dependent on accurate protein-protein interactions. Therefore, co-factors for c-Jun may define the functions for c-Jun in cancer. Identification of protein-protein interactions promoting cancer may provide novel possibilities for cancer treatment. In this study, we show that DNA topoisomerase I (TopoI) is a transcriptional co-factor for c-Jun. Moreover, c-Jun and TopoI together promote expression of epidermal growth factor receptor (EGFR) in cancer cells. We also show that the clinically used TopoI inhibitor topotecan reduces EGFR expression. Importantly, the effect of TopoI on EGFR transcription was shown to depend on c-Jun as Jun-/- cells or cells treated with JNK inhibitor SP600125 are resistant to topotecan treatment both in regulation of EGFR expression and cell proliferation. Moreover, c-Jun regulates the nucleolar localization and the function of the ribonucleic acid (RNA) helicase DDX21, a previously identified member of c-Jun protein complex. In addition, c-Jun stimulates rRNA processing by supporting DDX21 rRNA binding. Finally, this study characterizes a DDX21 dependent expression of cyclin dependent kinase (Cdk) 6, a correlation of DDX21 expression with prostate cancer progression and a substrate binding dependency of DDX21 nucleolar localization in prostate cancer cells. Taken together, the results of this study validate the c-Jun-TopoI interaction and precise the c-Jun-DDX21 interaction. Moreover, these results show the importance for protein-protein interaction in the regulation of their cellular functions in cancer cell behavior. Finally, the results presented here disclose new exciting therapeutic opportunities for cancer treatment.

The Integrin Tail: A Tale of Cell Motility and Division

Integrin transmembrane receptor functions are regulated by adaptor molecules binding to their alpha and beta subunit intracellular domains, or tails, thus affecting integrin traffic and adhesion during e.g. cell motility. Interestingly, many cellular proteins function in both cell motility and cell division, thus raising the possibility that integrins might be involved in regulating the cell cycle. A thorough understanding of cell division is essential in cell biology and in human malignancies. It is well established that failures to complete cell cycle can give rise to genetically unstable cells with tumorigenic properties. Transformed cells promote the disruption of intercellular adhesions such as tight junctions, and this correlates with the onset of cell motility, invasion and unfavorable prognosis in cancer. In this study, we analyzed integrin regulation, mediated by adaptor binding to the  subunit tail, during cell motility and cell division. We revealed a novel molecular mechanism by which Rab21, through association with the integrin alpha subunits, drives integrin endosomal traffic during mitotic phases. In addition, we found indications for this finding in vivo, as RAB21 gene deletions were mapped in ovarian and prostate cancer samples. Importantly, the multinucleated phenotype of cultured ovarian cancer cells could be reverted by Rab21 overexpression. In this thesis work, we also show how the tight junction protein ZO-1 unexpectedly interacts with the 5 integrin cytoplasmic domain in the lamellipodia to promote cell motility and at the cleavage furrow to support separation of the daughter cells. The alpha5-ZO-1 complex formation was dependent on PKC which regulates ZO-1 phosphorylation and its subcellular localization. In addition, by an in situ detection method, we showed that a subset of metastatic human lung cancers expressed the alpha5beta-ZO-1 complex. Taken together, we were able to identify new molecular pathways that regulate integrin functions in an alpha tail-mediated fashion. These findings firmly suggest that genetic alterations in integrin traffic may lead to progression of tumorigenesis as a result of failed cell division. Also, the interplay of integrins and ZO-1 in forming spatially regulated adhesive structures broadens our view of crosstalk between pathways and distinct adhesive structures that can be involved in cancer cell biology.

Regulation of B Cell Gene Expression and Function by Ikaros, Helios and Bcl6

B lymphocytes constitute a key branch of adaptive immunity by providing specificity to recognize a vast variety of antigens by B cell antigen receptors (BCR) and secreted antibodies. Antigen recognition activates the cells and can produce antibody secreting plasma cells via germinal center reaction that leads to the maturation of antigen recognition affinity and switching of antibody effector class. The specificity of antigen recognition is achieved through a multistep developmental pathway that is organized by interplay of transcription factors and signals through BCR. Lymphoid malignancies arise from different stages of development in abnormal function of transcriptional regulation. To understand the B cell development and the function of B cells, a thorough understanding of the regulation of gene expression is important. The transcription factors of the Ikaros family and Bcl6 are frequently associated with lymphoma generation. The aim of this study was to reveal the targets of Ikaros, Helios and Bcl6 mediated gene regulation and to find out the function of Ikaros and Helios in B cells. This study uses gene targeted DT40 B cell lines and establishes a role for Ikaros family factors Ikaros and Helios in the regulation of BCR signaling that is important at developmental checkpoints, for cell survival and in activation. Ikaros and Helios had opposing roles in the regulation of BCR signals. Ikaros was found to directly repress the SHIP gene that encodes a signaling lipid-metabolizing enzyme, whereas Helios had activating effect on SHIP expression. The findings demonstrate a balancing function for these two Ikaros family transcription factors in the regulation of BCR signaling as well as in the regulation of gene expression. Bcl6 was found to repress plasma cell gene expression program while maintaining gene expression profile of B cells. Analysis of direct Bcl6 target genes suggested novel mechanisms for Bcl6-mediated suppression of plasma cell differentiation and promoting germinal center phenotype.

The regulation and function of collagenase-3 (MMP-13) in cutaneous wound healing and squamous cell carcinoma

Matrix metalloproteinase-13 (MMP-13) is a potent proteolytic enzyme, whose expression has been previously associated with fetal bone development and postnatal bone remodeling and with adult gingival wound healing. MMP-13 is also known to be involved in the growth and invasion of various cancers including squamous cell carcinoma (SCC) of the skin. The aim of this study was to further elucidate the function and regulation of MMP-13 in wound repair and cancer. In this study, it was shown that fetal skin fibroblasts express MMP-13 in response to transforming growth factor-β in a p38 MAP kinase dependent manner. In addition, MMP-13 was found to be expressed in vivo by wound fibroblasts in human fetal skin grafted on SCID mice. Adenovirally delivered expression of MMP-13 enhanced collagen matrix contraction by fibroblasts in vitro in association with altered cytoskeletal structure, enhanced proliferation and survival. These results indicate that MMP-13 is involved in cell-mediated collagen matrix remodeling and suggest a role for MMP-13 in superior matrix remodeling and scarless healing of fetal skin wounds. Using an MMP-13 deficient mouse strain, it was shown that MMP-13 is essential for the normal development of experimental granulation tissue in mice. MMP-13 was implicated in the regulation of myofibroblast function and angiogenesis and the expression of genes involved in cellular proliferation and movement, immune response, angiogenesis and proteolysis. Finally, epidermal mitogen, keratinocyte growth factor (KGF) was shown to suppress the malignant properties of skin SCC cells by downregulating the expression of several target genes with potential cancer promoting properties, including MMP-13, and by reducing SCC cell invasion. These results provide evidence that MMP-13 potently regulates cell viability, myofibroblast function and angiogenesis associated with wound healing and cancer. In addition, fibroblasts expressing MMP-13 show high collagen reorganization capacity. Moreover, the results suggest that KGF mediates the anti-cancer effects on skin SCC