Regulation of Hematopoietic Cell Development and Hair Growth in Mouse Models: Expression Analysis of Tudor-SN and Polyamine-Regulated Proteins

Abstract

Malliorganismeiksi kutsutaan eliöitä, joilla voidaan mallintaa ihmisessä tapahtuvia fysiologisia toimintoja ja sairauksia. Tässä väitöskirjatutkimuksessa käytettiin hiirimallia selvitettäessä Tudor-SN ja polyamiini-säädeltyjen proteiinien ilmentymistä ja toimintaa.

Tudor-SN proteiinille on ehdotettu useita mekanismeja geenien ilmentymisen säätelijänä. Kuitenkaan itse Tudor-SN proteiinin ilmentymistä eri kudoksissa ei ole tarkoin tutkittu ja proteiinin fysiologinen merkitys on epäselvä. Tutkimuksessa selvisi, että Tudor-SN ilmentyy erityisesti nopeasti jakaantuvissa soluissa ja erilaistuvien solutyyppien jakaantuvissa esiastesoluissa. Tudor-SN poistogeenisen hiiren analyysissä todettiin sekä luuytimessä että verenkierrossa alentunut granulosyytti-solujen granulaarisuus sekä alentunut granulosyyttien määrä verenkierrossa. Tulokset ehdottavat, että Tudor-SN proteiini on merkityksellinen immuunijärjestelmässä.

Luonnon polyamiineista putreskiini, spermidiini ja spermiini ovat ratkaisevia nisäkkäiden solujen erilaistumisessa ja lisääntymisessä. Tutkimuksessa selvitettiin polyamiinien vaikutusta karvan kasvuun, joka tapahtuu kolmessa eri vaiheessa: nopea solujakaantuminen (anagen), apoptoosi (catagen) ja loppuvaiheen lepotila (telogen). Anagen-vaiheessa olevissa karvasoluissa on raportoitu korkea polyamiinipitoisuus. Tässä tutkimuksessa selvitettiin metabolisesti stabiilin polyymiinianalogin, alpha-metyylispermidiinin vaikutus karvan kasvuun. Kahden viikon päivittäinen alpha-metyylispermidiini annostelu telogen-vaiheessa oleville hiirillä aikaansai uudelleen nopean solujakaantumisen, eli anagen-vaiheen. Tulosten mukaan polyamiineilla voidaan vaikuttaa karvan kasvuun hiirimallissa.

Model organisms or reference organisms are non-human species, which are used to explore a given biological phenomenon deliberately to deduce the anticipated experimental findings to other organisms particularly to humans. Hypothetically, a given organism can be considered as a model organism when its size is small and it is competent to represent a certain specific organism by predicting myriads of biological and molecular processes related to genetics, development, physiology, evolution and ecology. By taking genetic conservation into account, a model organism with small size or simple form of life is expected to represent a larger organism including humans with complex genome and biological processes. Although no single organisms fulfill these theoretical criteria, most of our current knowledge of heredity, development, physiology and underlining molecular and cellular processes is obtained from studies based on model organisms. In this study, by using mouse as a model organism expression of Tudor-SN and polyamine regulated proteins were analyzed.

Tudor-SN staphylococcal nuclease (Tudor-SN) is a 100 kDa protein that was initially identified as a transcriptional co-activator. It has also been shown to function as a modulator of RNA metabolism and biogenesis and a component in the RNA-induced silencing complex (RISC). Tudor-SN protein is highly conserved through evolution from unicellular organisms such as yeast to higher organisms and it exists as a single gene without any close homologs. However, comprehensive analysis of the expression of Tudor-SN has not been investigated and the physiological function of Tudor-SN is poorly understood. In this study the expression of Tudor-SN was investigated in mouse tissues and organs by immunohistochemistry, fluorescent immunostaining, Western blotting and RT-qPCR. Up-regulated or high level expression of Tudor-SN was observed in rapidly dividing and progenitor cells such as in spermatogonial cells of testis, in the follicular cells of ovary, in the cells of crypts of Lieberkühn of ileum and basal keratinocytes of skin and hair follicle when compared to more differentiated or terminally differentiated cells in the respective organs. Analysis of Tudor-SN knockout bone marrow and peripheral blood cells indicated decrease in granularity of granulocytes. The wide expression pattern of Tudor-SN and high expression in proliferating and self-differentiating cells suggests that the protein serves functions related to activated state of cells. Besides, decreases in the numbers of eosinophils and granularity in bone marrow granulocytic cells in Tudor-SN knockout mouse suggests that the protein has an important role in immune system.

Natural polyamines putrescine, spermidine and spermine are crucial for mammalian cell differentiation and proliferation. In mammals, the growth of hair is characterized by three main cyclic phases of transformation, including a rapid growth phase (anagen), an apoptosis-driven regression phase (catagen) and a relatively quiescent resting phase (telogen). A high pool of polyamines is reported during the anagen phase compared to telogen and catagen phases. Hair cycle-associated fluctuation of polyamine concentrations and the skin abnormalities in the SSAT transgenic animals prompted us to investigate whether the growth of hair during the resting stage could be triggered by artificial elevation of the polyamine pool. However, natural polyamines are not stable molecules. α-Methylspermidine, a metabolically stable polyamine analog, has been used as surrogate molecule to elucidate the specific role of polyamines. The application of α-Methylspermidine in telogen mice induced hair growth after 2 weeks of daily topical application. The application site was characterized by typical features of anagen and the result suggests that polyamines play an important role in the regulation of hair cycle.