ÂÇÀÈÌÍÎÅ ÂËÈßÍÈÅ ð53/ð21/Rb È ÌÀÐ-ÊÈÍÀÇÍÛÕ ÑÈÃÍÀËÜÍÛÕ ÏÓÒÅÉ
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2015 ÖÈÒÎËÎÃÈß Ò î ì 57, ¹ 11 ÂÇÀÈÌÍÎÅ ÂËÈßÍÈÅ ð53/ð21/Rb È ÌÀÐ-ÊÈÍÀÇÍÛÕ ÑÈÃÍÀËÜÍÛÕ ÏÓÒÅÉ Â ÝÍÄÎÌÅÒÐÈÀËÜÍÛÕ ÑÒÂÎËÎÂÛÕ ÊËÅÒÊÀÕ ×ÅËÎÂÅÊÀ  ÓÑËÎÂÈßÕ ÎÊÈÑËÈÒÅËÜÍÎÃÎ ÑÒÐÅÑÑÀ © Ï. È. Äåðÿáèí, À. Â. Áîðîäêèíà, Í. Í. Íèêîëüñêèé, Å. Á. Áóðîâà1 Èíñòèòóò öèòîëîãèè ÐÀÍ, Ñàíêò-Ïåòåðáóðã, 194064; 1 ýëåêòðîííûé àäðåñ: [email protected] Ï. È. Äåðÿáèí è äð. Íåäàâíî ìû ïîêàçàëè, ÷òî â ìåçåíõèìíûõ ñòâîëîâûõ êëåòêàõ ýíäîìåòðèÿ ÷åëîâåêà (ÑÊÝ) â îòâåò íà îêèñëèòåëüíûé ñòðåññ àêòèâèðóþòñÿ ñèãíàëüíûå ïóòè ð53/p21/Rb è ð38/MAPKAPK-2, èãðàþùèå îïðåäåëÿþùóþ ðîëü â èíäóêöèè ïðåæäåâðåìåííîãî ñòàðåíèÿ ÑÊÝ. Îäíàêî äî ñèõ ïîð íå áûëî ïðîâåäåíî êîìïëåêñíîãî àíàëèçà âçàèìîäåéñòâèÿ ìåæäó ð53/p21/Rb è ñåìåéñòâîì ìèòîãåíàêòèâèðóåìûõ ïðîòåèíêèíàç (MAP-êèíàç), âêëþ÷àþùèì â ñåáÿ ð38, ERK1/2 è JNK.  íàñòîÿùåé ðàáîòå ìû èçó÷èëè ôóíêöèîíàëüíóþ àêòèâíîñòü ð53/p21/Rb è MAP-êèíàçíûõ ñèãíàëüíûõ ïóòåé â óñëîâèÿõ îêèñëèòåëüíîãî ñòðåññà ïðè èíãèáèðîâàíèè âõîäÿùèõ â íèõ êîìïîíåíòîâ. Ñîãëàñíî ðåçóëüòàòàì èíãèáèòîðíîãî àíàëèçà, ïðîâåäåííîãî ñ èñïîëüçîâàíèåì ñïåöèôè÷íûõ èíãèáèòîðîâ ÌÀÐ-êèíàç è ð53, ñóïðåññèÿ êàæäîé èç ÌÀÐ-êèíàç â Í2Î2-ñòèìóëèðîâàííûõ êëåòêàõ â çíà÷èòåëüíîé ñòåïåíè óñèëèâàëà àêòèâàöèþ ð53, à òàêæå ïðèâîäèëà ê ïîâûøåíèþ ýêñïðåññèè áåëêà ð21 è ñîîòâåòñòâåííî ãèïîôîñôîðèëèðîâàíèþ Rb.  òî æå âðåìÿ â ðåçóëüòàòå èíãèáèðîâàíèÿ ð53 ïîâûøàëñÿ óðîâåíü àêòèâàöèè âñåõ ÌÀÐ-êèíàç, íî â íàèáîëüøåé ñòåïåíè ERK1/2. Ïîëó÷åííûå ðåçóëüòàòû ïîçâîëÿþò ïðåäïîëàãàòü íàëè÷èå âçàèìíîé íåãàòèâíîé ðåãóëÿöèè ìåæäó ð53-çàâèñèìûìè è ÌÀÐ-êèíàçíûìè ñèãíàëüíûìè ïóòÿìè. Áûëî ïîêàçàíî òàêæå, ÷òî â Í2Î2-ñòèìóëèðîâàííûõ ÑÊÝ ñóùåñòâóåò ñëîæíîîðãàíèçîâàííàÿ ñèñòåìà âçàèìîäåéñòâèé ìåæäó ÷ëåíàìè ñåìåéñòâà ÌÀÐ-êèíàç. Êàê îêàçàëîñü, ÌÀÐ-êèíàçû ð38 è JNK ôóíêöèîíèðóþò â êà÷åñòâå àíòàãîíèñòîâ â ðåãóëÿöèè ERK1/2 : åñëè JNK àêòèâèðóåò, òî ð38 áëîêèðóåò àêòèâàöèþ ERK1/2. Ñ äðóãîé ñòîðîíû, èíãèáèðîâàíèå ERK1/2 ïîíèæàëî àêòèâíîñòü è ð38, è JNK. Íàñòîÿùàÿ ðàáîòà óñòàíàâëèâàåò ñëîæíóþ âçàèìîñâÿçü ìåæäó àêòèâíîñòüþ ð53/ð21/Rb è êàæäûì èç òðåõ ÌÀÐ-êèíàçíûõ ïóòåé è îòêðûâàåò íîâûå íàïðàâëåíèÿ èññëåäîâàíèé ôåíîìåíà ïðåæäåâðåìåííîãî ñòàðåíèÿ. Âçàèìíîå âëèÿíèå p53/p21/Rb è MAP-êèíàçíûõ ñèãíàëüíûõ ïóòåé Ê ë þ ÷ å â û å ñ ë î â à: ñòâîëîâûå êëåòêè, îêèñëèòåëüíûé ñòðåññ, ÌÀÐ-êèíàçû, ð53, ïèôèòðèí-a, èíãèáèòîðíûé àíàëèç. Ï ð è í ÿ ò û å ñ î ê ð à ù å í è ÿ: ÑÊÝ — ñòâîëîâûå êëåòêè ýíäîìåòðèÿ, JNK — c-Jun N-êîíöåâàÿ êèíàçà (c-Jun N-terminal kinase), ERK — êèíàçà, ðåãóëèðóåìàÿ ýêñòðàêëåòî÷íûìè ñèãíàëàìè (extracellular signal-regulated kinase), TNF-a — ôàêòîð íåêðîçà îïóõîëåé-a, IL-1b — èíòåðëåéêèí-1b, ASK — àïîïòîçðåãóëèðóþùàÿ êèíàçà (apoptosis signal-regulating kinase), DDR 1 — discoidin domain receptor 1, Rb — áåëîê ðåòèíîáëàñòîìû, MKP-3 — ôîñôàòàçà 3 ÌÀÐ-êèíàçû (MAP kinase phosphatase 3), ÌÀÐKÀÐK-2 (èëè ÌK-2) — ïðîòåèíêèíàçà 2, àêòèâèðóåìàÿ ÌÀÐ-êèíàçîé (MAP kinase-activated protein kinase 2). Èíôîðìàöèîííîå ïðîñòðàíñòâî êëåòêè, ñîäåðæàùåå ñëîæíîîðãàíèçîâàííóþ ñåòü ñèãíàëüíûõ ïóòåé, ôóíêöèîíèðóþùèõ ïî ïðèíöèïó ïðÿìîé è îáðàòíîé ñâÿçè, â êà÷åñòâå íåïðåìåííîãî êîìïîíåíòà âêëþ÷àåò â ñåáÿ ìèòîãåíàêòèâèðóåìûå ïðîòåèíêèíàçíûå (ÌÀÐ-êèíàçíûå) êàñêàäû. Îñíîâíûìè ïðåäñòàâèòåëÿìè ñåìåéñòâà ÌÀÐêèíàç ÿâëÿþòñÿ ñòðåññàêòèâèðóåìûå ïðîòåèíêèíàçû JNK è ð38, à òàêæå ðåãóëèðóåìûå ýêñòðàêëåòî÷íûìè ñèãíàëàìè êèíàçû ERK1/2.  îòâåò íà âíåêëåòî÷íûå ñòèìóëû ÌÀÐ-êèíàçû êîîðäèíèðóþò øèðîêèé ñïåêòð âíóòðèêëåòî÷íîé àêòèâíîñòè — îò ìåòàáîëèçìà, ïîäâèæíîñòè, ìèòîçà, âîñïàëåíèÿ è äèôôåðåíöèðîâêè äî êëåòî÷íîé ãèáåëè è âûæèâàíèÿ (Wu, 2004; McCubrey et al., 2007; Kyriakis, Avruch, 2012; Kim, Choi, 2015). Ñ êàíîíè÷åñêîé òî÷êè çðåíèÿ, ERK1/2 àêòèâèðóþòñÿ ìèòîãåíàìè (ðîñòîâûìè ôàêòîðàìè, ôîðáîëîâûìè ýôèðàìè), îäíàêî áûëî ïîêàçàíî, ÷òî ñòðåññîâûå ôàêòîðû òàêæå ñïîñîáíû ñòèìóëèðî- âàòü ýòîò ïóòü (Lewis et al., 1998; Burova et al., 2007).  îòëè÷èå îò ERK, ñòðåññ-êèíàçû ð38 è JNK àêòèâèðóþòñÿ, êàê ïðàâèëî, ñòðåññàìè ðàçíûõ òèïîâ, âêëþ÷àÿ îêèñëèòåëüíûé, ãåíîòîêñè÷åñêèé, îñìîòè÷åñêèé, à òàêæå èíäóöèðîâàííûé ïðîâîñïàëèòåëüíûìè öèòîêèíàìè TNF-a è IL-1b (Kyriakis, Avruch, 2012). Ïåðåäàþùèå ñèãíàë ÌÀÐ-êèíàçíûå êàñêàäû îðãàíèçîâàíû â âèäå ìîäóëåé, ñîäåðæàùèõ èåðàðõè÷åñêóþ ïîñëåäîâàòåëüíîñòü ïðîòåèíêèíàç, àêòèâíîñòü êîòîðûõ ðåãóëèðóåòñÿ ïðè ïîìîùè ðàçëè÷íûõ, ñïåöèôè÷íûõ äëÿ êàæäîãî ìîäóëÿ êèíàç è ôîñôàòàç ïóòåì ôîñôîðèëèðîâàíèÿ èëè äåôîñôîðèëèðîâàíèÿ ïî ñåðèíîâûì, òðåîíèíîâûì è òèðîçèíîâûì îñòàòêàì. Ïîñëå ñòèìóëÿöèè ïðîèñõîäèò àêòèâàöèÿ êèíàçû ASK â ñëó÷àå JNK è ð38 (Fujisawa et al., 2007) èëè Raf ïîñðåäñòâîì GTP-ñâÿçûâàþùåãî áåëêà Ras â ñëó÷àå ERK (Chang, Karin, 2001; Pearson et al., 2001). Äàëåå ASK è Raf ñòèìóëèðóþò àêòèâíîñòü êèíàç 788 Âçàèìíîå âëèÿíèå p53/p21/Rb è MAP-êèíàçíûõ ñèãíàëüíûõ ïóòåé äâîéíîé ñïåöèôè÷íîñòè (êèíàçû ÌÀÐ-êèíàç èëè ÌKK), êîòîðûå âêëþ÷àþò â ñåáÿ ÌKK-1/2 (èëè ÌÅK1/2), ÌKK-3/6 è ÌKK-4/7, ñåëåêòèâíî àêòèâèðóþùèå ERK1/2, ð38 è JNK ñîîòâåòñòâåííî (Kyriakis, Avruch, 2012). Àêòèâèðîâàííûå ÌÀÐ-êèíàçû ôîñôîðèëèðóþò ðàçëè÷íûå áåëêè-ìèøåíè, âêëþ÷àÿ òðàíñêðèïöèîííûå ôàêòîðû c-Jun, AP-1, p53, c-Myc, Elk1 è ATF2 íàðÿäó ñ àíòèàïîïòîòè÷åñêèìè è ïðîàïîïòîòè÷åñêèìè áåëêàìè Bcl-2 è Bad ñîîòâåòñòâåííî (Bogoyevitch, Kobe, 2006; Takeda et al., 2011). Êëàññè÷åñêèå ïðåäñòàâëåíèÿ, ñîãëàñíî êîòîðûì ERK ôóíêöèîíèðóåò ïðåèìóùåñòâåííî â ïåðåäà÷å ïðîëèôåðàòèâíîãî ñèãíàëà ñ ìåìáðàííî-ñâÿçàííûõ ðåöåïòîðîâ è îïîñðåäóåò äèôôåðåíöèðîâêó è âûæèâàíèå êëåòîê, à ð38 è JNK âîâëå÷åíû â èíèöèàöèþ êëåòî÷íîé ãèáåëè, ïîñòîÿííî ðàñøèðÿþòñÿ ñ ïîÿâëåíèåì íîâûõ äàííûõ, ñâèäåòåëüñòâóþùèõ î íåîäíîçíà÷íîñòè ôóíêöèîíàëüíîé ðîëè ÌÀÐ-êèíàç. Òàê, â çàâèñèìîñòè îò ñòèìóëà è êëåòî÷íîãî êîíòåêñòà Raf/ÌÅK/ERK-ïóòü âëèÿåò â ðàçíîé ñòåïåíè è äàæå ïðîòèâîðå÷èâûì îáðàçîì íà àïîïòîç, ñòàðåíèå, îñòàíîâêó êëåòî÷íîãî öèêëà è ïðîëèôåðàöèþ (McCubrey et al., 2007; Sawe et al., 2008). Àêòèâèðîâàííûå ðàçëè÷íûìè ñòðåññîâûìè ôàêòîðàìè ð38 è JNK ñèãíàëüíûå ïóòè âîâëå÷åíû íå òîëüêî â àïîïòîç (Xia et al., 1995; Porras et al., 2004; Guan et al., 2006; Xiao et al., 2015), íî è â ñòàðåíèå êëåòîê (Zarubin, Han, 2005; Debacq-Chainiaux et al., 2010; Passos et al., 2010; Borodkina et al., 2014). Òðàíñêðèïöèîííûé ôàêòîð ð53, ïðîÿâëÿþùèé òóìîð-ñóïðåññîðíóþ àêòèâíîñòü, â îòâåò íà ãåíîòîêñè÷åñêèé ñòðåññ ðåãóëèðóåò ýêñïðåññèþ ðàçëè÷íûõ ãåíîâ, èíäóöèðóþùèõ ìåõàíèçìû àíòèêàíöåðîãåíåçà, âêëþ÷àÿ àðåñò êëåòî÷íîãî öèêëà, àïîïòîç è ñòàðåíèå, â çàâèñèìîñòè îò êëåòî÷íîãî êîíòåêñòà, òèïà è æåñòêîñòè ñòðåññà (Wu, 2004; Riley et al., 2008; Menendez et al., 2009). Òðàíñêðèïöèîííàÿ àêòèâíîñòü ð53 ïðîÿâëÿåòñÿ â ðåçóëüòàòå âçàèìîäåéñòâèÿ ñ ìíîãî÷èñëåííûìè ñèãíàëüíûìè ïóòÿìè, â òîì ÷èñëå ÌÀÐ-êèíàçíûìè — p38 (She et al., 2001; Duan et al., 2011; Xiao et al., 2015), JNK (Wu, 2004; Hsu et al., 2009; Lorin et al., 2010) è ERK (Persons et al., 2000; Kaji et al., 2003; Lin et al., 2008; Drosten et al., 2014), êîòîðûå ôîñôîðèëèðóþò è àêòèâèðóþò ð53 ïðè ñòèìóëÿöèè ñòðåññîâûìè ôàêòîðàìè.  ñâîþ î÷åðåäü ð53 ìîæåò àêòèâèðîâàòü Ras/Raf/ÌÅK/ERK-ñèãíàëèíã (Wu, 2004; Singh et al., 2007; Lee et al., 2013) ÷åðåç ìåõàíèçì, îïîñðåäîâàííûé ð53-çàâèñèìîé èíäóêöèåé òèðîçèíêèíàçíîãî ðåöåïòîðà DDR 1 (Ongusaha et al., 2003) è ëèãàíäîâ ðåöåïòîðà ýïèäåðìàëüíîãî ôàêòîðà ðîñòà (Lee et al., 2000; Fang et al., 2001). Ïðåäïîëàãàåòñÿ, ÷òî òàêîé ìåõàíèçì èãðàåò âàæíóþ ðîëü â çàùèòå ðàêîâûõ êëåòîê îò ð53-çàâèñèìîãî àïîïòîçà.  òî æå âðåìÿ òðàíñêðèïöèîííûé ôàêòîð ð53 ñïîñîáåí ïîäàâëÿòü àêòèâíîñòü ERK, àêòèâèðóÿ ãåíû ðÿäà ôîñôàòàç, êîòîðûå äåôîñôîðèëèðóþò ERK è áëîêèðóþò åå àíòèàïîïòîòè÷åñêóþ àêòèâíîñòü, ÷òî ìîæåò ñïîñîáñòâîâàòü ð53-èíäóöèðîâàííîìó àïîïòîçó (Bermudez et al., 2011; Zhang et al., 2015). Î÷åâèäíî, ÷òî êîíå÷íûé îòâåò êëåòîê îïðåäåëÿåòñÿ áàëàíñîì ìåæäó ðàçíîîáðàçíûìè ñòðåññ-àêòèâèðîâàííûìè ïóòÿìè. Ïîìèìî èíäóêöèè àïîïòîçà ð53 èãðàåò îïðåäåëÿþùóþ ðîëü â ðåïëèêàòèâíîì è ñòðåññ-èíäóöèðîâàííîì ñòàðåíèè êëåòîê ðàçíûõ òèïîâ, íåîáðàòèìî èíãèáèðóÿ ïðîãðåññèþ êëåòî÷íîãî öèêëà â ñîñòàâå ð53/ð21/Rb-ñèãíàëüíîãî ïóòè, ÷òî ïðèâîäèò ê ïîòåðå ïðîëèôåðàòèâíîãî ïîòåíöèàëà, õàðàêòåðíîé, íàðÿäó ñ èçìåíåíèåì ôåíîòèïà, äëÿ ñòàðûõ êëåòîê.  ïðåäûäóùèõ èññëåäîâàíèÿõ ìû óñòàíîâèëè, ÷òî ìåçåíõèìíûå ñòâîëîâûå êëåòêè ýíäîìåò- 789 ðèÿ ÷åëîâåêà (ÑÊÝ) ïðåæäåâðåìåííî ñòàðåþò â óñëîâèÿõ ñóáëåòàëüíîãî îêèñëèòåëüíîãî ñòðåññà.  ïåðåäà÷ó îêèñëèòåëüíîãî ñèãíàëà, èíäóöèðóþùåãî ïðîöåññ ñòàðåíèÿ ÑÊÝ, âîâëå÷åíû ïóòè ð53/ð21/Rb è ð38/MAPKAPK-2 (Borodkina et al., 2014). Öåëüþ ïðåäñòàâëåííîé ðàáîòû ÿâëÿåòñÿ îöåíêà âçàèìíîãî âëèÿíèÿ ð53/ð21/Rb- è ÌÀÐ-êèíàçíûõ ñèãíàëüíûõ ïóòåé, âêëþ÷àþùèõ â ñåáÿ ERK, ð38 è JNK, ïðè îêèñëèòåëüíîì ñòðåññå â ÑÊÝ. Ìàòåðèàë è ìåòîäèêà Êóëüòèâèðîâàíèå ê ë å ò î ê. Ñòâîëîâûå ê ë å ò ê è ý í ä î ì å ò ð è ÿ ÷åëîâåêà (ÑÊÝ), ïîëó÷åííûå â Èíñòèòóòå öèòîëîãèè ÐÀÍ (Ñàíêò-Ïåòåðáóðã), âûäåëÿëè èç äåñêâàìèðîâàííîãî ýíäîìåòðèÿ ìåíñòðóàëüíîé êðîâè çäîðîâûõ äîíîðîâ (Çåìåëüêî è äð., 2011) è êóëüòèâèðîâàëè â ñðåäå DMEM/F12 (Gibco, ÑØÀ), ñîäåðæàùåé 10 % ýìáðèîíàëüíîé ñûâîðîòêè (HyClone, ÑØÀ), 1 % ãåíòàìèöèíà è 1 % ãëóòàìàêñà, â àòìîñôåðå 5 % ÑÎ2 ïðè 37 °Ñ âî ôëàêîíàõ 25 èëè 75 ñì2. Äëÿ ýêñïåðèìåíòîâ êëåòêè ðàññåâàëè ñ ïëîòíîñòüþ 15 òûñ. êë./ñì2 íà ÷àøêè äèàìåòðîì 35 ìì; èñïîëüçîâàëè êëåòêè 6—9-ãî ïàññàæåé. Î ê è ñ ë è ò å ë ü í û é ñ ò ð å ñ ñ âûçûâàëè äîáàâëåíèåì â áåññûâîðîòî÷íóþ ðîñòîâóþ ñðåäó Í2Î2 (äî êîíå÷íîé êîíöåíòðàöèè 200 ìêÌ), ðàñòâîð êîòîðîé ãîòîâèëè èç 30%-íîé Í2Î2 íåïîñðåäñòâåííî ïåðåä èñïîëüçîâàíèåì. Îáðàáîòêó êëåòîê ïðîâîäèëè â òå÷åíèå 30 ìèí èëè 1 ÷ ïðè 37 °Ñ â àòìîñôåðå 5 % ÑÎ2, ïîñëå ÷åãî êëåòêè äâàæäû ïðîìûâàëè áåññûâîðîòî÷íîé ñðåäîé è äàëåå êóëüòèâèðîâàëè â ñâåæåé ðîñòîâîé ñðåäå.  ýêñïåðèìåíòàõ èñïîëüçîâàëè ñëåäóþùèå ôàðìàêîëîãè÷åñêèå è í ã è á è ò î ð û : ïèôèòðèí-a — ñåëåêòèâíûé èíãèáèòîð òðàíñêðèïöèîííîãî ôàêòîðà ð53 (Komarov et al., 1999); âåùåñòâî U0126 (1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene) — âûñîêîñåëåêòèâíûé èíãèáèòîð êèíàç ÌÅK1/2, ðåãóëèðóþùèõ àêòèâíîñòü ERK1/2 (Favata et al., 1998); SB203580 (4-[4-(4-fluorophenyl)-2-(4-(methylsulfinylphenyl)-1H-imidazol-5-yl] pyridine) — ñïåöèôè÷åñêèé èíãèáèòîð ð38 ÌÀÐ-êèíàçíîãî ïóòè, ñâÿçûâàþùèéñÿ â ÀÒÔ-ñâÿçûâàþùåì êàðìàíå ìîëåêóëû ð38 è ïðåäîòâðàùàþùèé ôîñôîðèëèðîâàíèå ìèøåíåé ð38, â ÷àñòíîñòè êèíàçû ÌK-2, íî íå ïðåïÿòñòâóþùèé ôîñôîðèëèðîâàíèþ ñàìîé êèíàçû ð38 (Young et al., 1997); SP600125 (anthrapyrazolone) — ñïåöèôè÷åñêèé èíãèáèòîð JNK (Bennett et al., 2001). Ýêñïåðèìåíòû ïðîâîäèëè ïî ñëåäóþùåé ñõåìå: 1) êëåòêè ïðåäâàðèòåëüíî êóëüòèâèðîâàëè â ïðèñóòñòâèè 10 ìêM U0126, 5 ìêM SB203580 èëè 5 ìêM SP600125 â òå÷åíèå 1 ÷ ëèáî 50 ìêM ïèôèòðèíà â òå÷åíèå 2 ÷ ïðè 37 °Ñ â àòìîñôåðå 5 % ÑÎ2; 2) çàòåì êëåòêè ïîäâåðãàëè îêèñëèòåëüíîìó ñòðåññó â ïðèñóòñòâèè îäíîãî èç èíãèáèòîðîâ è äàëåå êóëüòèâèðîâàëè â ñâåæåé ðîñòîâîé ñðåäå (áåç Í2Î2) â òå÷åíèå 3 ñóò ïðè ïîñòîÿííîì ïðèñóòñòâèè îäíîãî èç èññëåäóåìûõ èíãèáèòîðîâ. È ì ì ó í î á ë î ò è í ã. Êëåòêè äâàæäû ïðîìûâàëè õîëîäíûì PBS è ëèçèðîâàëè â áóôåðå, ñîäåðæàùåì 50 ìÌ Òðèñ-HCl, 150 ìÌ NaCl, 1 ìÌ ÝÄÒÀ, 1 ìÌ ÝÃÒÀ, 10 % ãëèöåðèíà, 1 % Òðèòîíà Õ-100, 1 ìÌ Na3VO4, 1 ìÌ NaF, 0.5 ìÌ PMSF è êîêòåéëü èíãèáèòîðîâ ïðîòåàç (1 : 500; Sigma, ÑØÀ), â òå÷åíèå 10 ìèí íà ëüäó. Ïîñëå ëèçèñà êëåòêè ñîñêðåáàëè ñ ÷àøåê è öåíòðèôóãèðîâàëè â òå÷åíèå 15 ìèí ïðè 15 000 g. Ê ñóïåðíàòàíòó äîáàâëÿëè 1/4 ÷àñòü áóôåðà äëÿ ýëåêòðîôîðåòè÷åñêèõ ïðîá (40 ìÌ 790 Ï. È. Äåðÿáèí è äð. Òðèñ, ðÍ 6.8, 10 % SDS, 20 % 2-ìåðêàïòîýòàíîëà è 40 % ãëèöåðèíà) è èíêóáèðîâàëè â òå÷åíèå 5 ìèí ïðè 100 °Ñ. Êîíöåíòðàöèþ áåëêà îïðåäåëÿëè ïî ìåòîäó Áðåäôîðä, èñïîëüçóÿ îâàëüáóìèí äëÿ ïîñòðîåíèÿ êàëèáðîâî÷íîé êðèâîé. Ýëåêòðîôîðåòè÷åñêîå ðàçäåëåíèå áåëêîâ ïðîâîäèëè ìåòîäîì SDS-ýëåêòðîôîðåçà â ïîëèàêðèëàìèäíîì ãåëå ñ ïîñëåäóþùèì ïåðåíîñîì áåëêîâ íà íèòðîöåëëþëîçíóþ ìåìáðàíó Hybond-C extra (Amersham Pharmacia Biotech, Øâåöèÿ). Äëÿ âèçóàëèçàöèè áåëêîâûõ ïîëîñ èñïîëüçîâàëè Ponceau S (Sigma, ÑØÀ). Äëÿ ñïåöèôè÷åñêîãî âûÿâëåíèÿ áåëêîâ èñïîëüçîâàëè ìîíîêëîíàëüíûå êðîëè÷üè àíòèòåëà ïðîòèâ èíãèáèòîðà öèêëèíçàâèñèìûõ êèíàç ð21Cip1, ãëèöåðàëüäåãèä-3-ôîñôàòäåãèäðîãåíàçû (GAPDH, clone 14C10), ôîñôî-ÌÀÐKÀÐK-2 (Thr334), ð38-a ÌÀÐK, SAPK/JNK, à òàêæå ïîëèêëîíàëüíûå êðîëè÷üè àíòèòåëà ïðîòèâ ôîñôî-Rb (Ser807/811), ôîñôî-ð53 (Ser15), ôîñôî-ð38 (Thr180/Tyr182), ERK2, ôîñôî-ERK1/2 (Thr202/Tyr204), ôîñôî-SAPK/JNK (Thr183/Tyr185) è p53.  êà÷åñòâå âòîðè÷íûõ àíòèòåë ïðèìåíÿëè êîçüè àíòèòåëà, âûðàáîòàííûå ïðîòèâ èììóíîãëîáóëèíîâ êðîëèêà, êîíúþãèðîâàííûå ñ ïåðîêñèäàçîé õðåíà (GAR-HRP). Âñå àíòèòåëà, çà èñêëþ÷åíèåì àíòèòåë ïðîòèâ ERK2 (Santa Cruz Biotechnology, ÑØÀ), áûëè ïîëó÷åíû îò ôèðìû Cell Signaling (ÑØÀ). Äëÿ äåòåêöèè ïåðîêñèäàçíîé àêòèâíîñòè êîíúþãàòîâ GAR-HRP èñïîëüçîâàëè ðåàêöèþ óñèëåííîé õåìèëþìèíåñöåíöèè (ECL, Amersham, Øâåöèÿ). Õåìèëþìèíåñöåíòíîå èçëó÷åíèå ðåãèñòðèðîâàëè ýêñïîíèðîâàíèåì íà ðåíòãåíîâñêóþ ïëåíêó CEA RP NEW (CEA AB, Øâåöèÿ).  ðàáîòå èñïîëüçîâàëè íåîðãàíè÷åñêèå ñîëè è ïåðåêèñü âîäîðîäà ïðîèçâîäñòâà ôèðìû Sigma (ÑØÀ). Ðåçóëüòàòû è îáñóæäåíèå  ïðåäûäóùèõ èññëåäîâàíèÿõ ìû óñòàíîâèëè, ÷òî ÑÊÝ ïðè ïóëüñîâîì âîçäåéñòâèè Í2Î2 â ñóáëåòàëüíûõ êîíöåíòðàöèÿõ âõîäÿò â ñîñòîÿíèå ïðåæäåâðåìåííîãî ñòàðåíèÿ, êîòîðîå ïîñòåïåííî ðàçâèâàåòñÿ âî âðåìåíè è ñòàíîâèòñÿ íåîáðàòèìûì (Burova et al., 2013). Àêòèâàöèÿ îñíîâíûõ ñåíñîðîâ îêèñëèòåëüíîãî âîçäåéñòâèÿ, âêëþ÷àÿ êèíàçó ÀÒÌ, ãèñòîí H2AX, àäàïòîðíûé áåëîê 53ÂÐ1 è òðàíñêðèïöèîííûé ôàêòîð ð53, ïðîèñõîäèò â òå÷åíèå ïåðâûõ 5—30 ìèí ñòèìóëÿöèè, òîãäà êàê íåîáðàòèìîå ñòàðåíèå ÑÊÝ íàñòóïàåò êàê ìèíèìóì ÷åðåç 1—2 ñóò (Borodkina et al., 2014).  îñíîâó èçó÷åíèÿ âçàèìîäåéñòâèÿ ÌÀÐ-êèíàçíûõ è ð53-çàâèñèìûõ ñèãíàëüíûõ ïóòåé áûë ïîëîæåí êîìïëåêñíûé èíãèáèòîðíûé àíàëèç ñ èñïîëüçîâàíèåì ñïåöèôè÷åñêèõ èíãèáèòîðîâ U0126, SB203580, SP600125 è ïèôèòðèíà-a äëÿ áëîêèðîâàíèÿ àêòèâíîñòè áåëêîâûõ êîìïîíåíòîâ ýòèõ ïóòåé — ERK1/2, p38, JNK è p53 ñîîòâåòñòâåííî. Èçìåíåíèå ñòàòóñà ôîñôîðèëèðîâàíèÿ áåëêîâ â îòâåò íà äåéñòâèå Í2Î2 â ïðèñóòñòâèè èíãèáèòîðîâ àíàëèçèðîâàëè ìåòîäîì Âåñòåðí-áëîòèíãà â äâóõ âðåìåíí*ûõ òî÷êàõ: ÷åðåç 30 ìèí (èíèöèàöèÿ èíäóöèðîâàííîãî ñòàðåíèÿ) è ÷åðåç 3 ñóò ïîñëå äåéñòâèÿ Í2Î2 (ôàçà íåîáðàòèìîãî èíäóöèðîâàííîãî ñòàðåíèÿ). Ðèñ. 1. Ñòàòóñ ôîñôîðèëèðîâàíèÿ MAP-êèíàç è òðàíñêðèïöèîííîãî ôàêòîðà p53 ïîñëå 30-ìèíóòíîãî äåéñòâèÿ H2O2 íà ìåçåíõèìíûå ñòâîëîâûå êëåòêè ýíäîìåòðèÿ (ÑÊÝ) ÷åëîâåêà â ïðèñóòñòâèè è â îòñóòñòâèå èõ èíãèáèòîðîâ. Èñïîëüçîâàëè ñïåöèôè÷åñêèå èíãèáèòîðû p53 (ïèôèòðèí-a: PFT) è êèíàç p38 (SB203580: SB), JNK (SP600125: SP) è ERK1/2 (U0126: U). Óðîâåíü ôîñôîðèëèðîâàíèÿ p53 è MAP-êèíàç îïðåäåëÿëè â èììóíîáëîòå ñ ïîìîùüþ àíòèòåë, ñïåöèôè÷íûõ ê èõ ôîñôîðèëèðîâàííûì ôîðìàì (ñì. ðàçäåë «Ìàòåðèàë è ìåòîäèêà»). Ê — êîíòðîëü. ×èñëà ïîêàçûâàþò ðåçóëüòàòû äåíñèòîìåòðèè, íîðìèðîâàííûå ê êîíòðîëþ. Âçàèìíîå âëèÿíèå p53/p21/Rb è MAP-êèíàçíûõ ñèãíàëüíûõ ïóòåé 791 Ðèñ. 2. Ìîäóëÿöèÿ óðîâíÿ H2O2-èíäóöèðîâàííîãî ôîñôîðèëèðîâàíèÿ è ýêñïðåññèè MAP-êèíàç è p53 ïðè äåéñòâèè ñïåöèôè÷åñêèõ èíãèáèòîðîâ. Ïîñëå îáðàáîòêè êëåòîê èíãèáèòîðàìè è H2O2 (â òå÷åíèå 1 ÷) êëåòêè êóëüòèâèðîâàëè â ïðèñóòñòâèè èíãèáèòîðîâ â òå÷åíèå 3 ñóò, ëèçèðîâàëè è àíàëèçèðîâàëè ìåòîäîì Âåñòåðí-áëîòèíãà. Ê — íåîáðàáîòàííûå êëåòêè. ×èñëà ïîêàçûâàþò ðåçóëüòàòû äåíñèòîìåòðèè ïîëîñ, íîðìèðîâàííûå ê êîíòðîëþ. Îñòàëüíûå îáúÿñíåíèÿ ñì. â ïîäïèñè ê ðèñ. 1. Ä å é ñ ò â è å ï è ô è ò ð è í à-a. Ïèôèòðèí ñåëåêòèâíî èíãèáèðóåò ð53, ïðåïÿòñòâóÿ åãî òðàíñëîêàöèè â ÿäðî, è ïîäàâëÿåò åãî òðàíñêðèïöèîííóþ àêòèâíîñòü, íî íå âëèÿåò íà ôîñôîðèëèðîâàíèå îñíîâíûõ ñàéòîâ ìîëåêóëû ð53, âêëþ÷àÿ Ser15 (Gudkov, Komarova, 2005). Ïðåäîáðàáîòêà ÑÊÝ ïèôèòðèíîì íå èçìåíÿëà óðîâåíü Í2Î2-èíäóöèðîâàííîãî ôîñôîðèëèðîâàíèÿ áåëêà ð53 ïî Ser15 (ðèñ. 1, 2), íî îêàçûâàëà ñóùåñòâåííîå âëèÿíèå êàê íà ýêñïðåññèþ áåëêà ð21, òðàíñêðèïöèîííóþ ìèøåíü ð53, òàê è íà ôîñôîðèëèðîâàíèå áåëêà Rb (ðèñ. 3, à). Ñëåäîâàòåëüíî, ïèôèòðèí ýôôåêòèâíî èíãèáèðóåò òðàíñêðèïöèîííóþ àêòèâíîñòü ð53 â Í2Î2-ñòèìóëèðîâàííûõ ÑÊÝ. Ðàíåå ìû ïðîäåìîíñòðèðîâàëè, ÷òî ïîâûøåíèå ýêñïðåññèè ð21 íåîáõîäèìî äëÿ çàïóñêà ïðåæäåâðåìåííîãî ñòàðåíèÿ â ÑÊÝ 792 Ï. È. Äåðÿáèí è äð. Ðèñ. 3. Âëèÿíèå èíãèáèðîâàíèÿ p53 è MAP-êèíàç íà óðîâåíü H2O2-èíäóöèðîâàííîãî ôîñôîðèëèðîâàíèÿ Rb è MK-2, à òàêæå ýêñïðåññèè áåëêà p21. Îáðàáîòêó ÑÊÝ ïðîâîäèëè, êàê óêàçàíî â ïîäïèñè ê ðèñ. 2. Ñ ïîìîùüþ ñïåöèôè÷íûõ àíòèòåë â èììóíîáëîòå âûÿâëÿëè: à — ôîñôîðèëèðîâàííóþ ôîðìó áåëêà Rb (pRb) è óðîâåíü ýêñïðåññèè p21 (p21Cip1); á — ôîñôîðèëèðîâàííóþ ôîðìó êèíàçû MK-2 (pMK-2). ×èñëà ïîêàçûâàþò ðåçóëüòàòû äåíñèòîìåòðèè ïîëîñ, íîðìèðîâàííûõ ê êîíòðîëþ. Ê — íåîáðàáîòàííûå êëåòêè. â îòâåò íà îêèñëèòåëüíûé ñòðåññ, âñëåäñòâèå ÷åãî áåëîê Rb ñòàíîâèòñÿ ãèïîôîñôîðèëèðîâàííûì (Borodkina et al., 2014).  êëåòêàõ, îáðàáîòàííûõ ïèôèòðèíîì, óðîâåíü ýêñïðåññèè ð21 ÷åðåç 3 ñóò áûë ñóùåñòâåííî ïîíèæåí ïî ñðàâíåíèþ ñ Í2Î2-ñòèìóëèðîâàííûìè êëåòêàìè, òîãäà êàê ôîñôîðèëèðîâàíèå Rb ïîâûøàëîñü â çíà÷èòåëüíîé ñòåïåíè. Ýòè ðåçóëüòàòû ñâèäåòåëüñòâóþò â ïîëüçó âîçìîæíîãî ïðåäîòâðàùåíèÿ ñòàðåíèÿ ÑÊÝ â óñëîâèÿõ áëîêèðîâàíèÿ ð53, êëþ÷åâîãî êîìïîíåíòà ð53/ð21/Rb-ïóòè, àêòèâèðóþùåãî ïðîãðàììó Í2Î2-èíäóöèðîâàííîãî ñòàðåíèÿ êëåòîê.  êîíòåêñòå âçàèìîäåéñòâèÿ ÌÀÐ-êèíàçíûõ è ð53/ð21/Rb-ñèãíàëüíûõ ïóòåé â ÑÊÝ áûëî èíòåðåñíî èñ- ñëåäîâàòü âëèÿíèå ïèôèòðèíà íà Í2Î2-èíäóöèðîâàííóþ àêòèâàöèþ êèíàç ERK1/2, p38 è JNK. Îêàçàëîñü, ÷òî ÷åðåç 30 ìèí äåéñòâèå ïèôèòðèíà ïðàêòè÷åñêè íå îòðàæàåòñÿ íà ôîñôîðèëèðîâàíèè ð38 è JNK, íî çíà÷èòåëüíî ïîâûøàåò óðîâåíü àêòèâàöèè ERK1/2 (ðèñ. 1). ×åðåç 3 ñóò ïèôèòðèí èíäóöèðóåò àêòèâàöèþ âñåõ ÌÀÐ-êèíàç, îäíàêî êèíàçû ERK1/2 ïî-ïðåæíåìó ñîõðàíÿëè ñàìóþ âûñîêóþ àêòèâíîñòü (ðèñ. 2).  ïðèñóòñòâèè ïèôèòðèíà óñèëèâàþòñÿ Í2Î2-èíäóöèðîâàííàÿ àêòèâàöèÿ ð38 è åå ïðÿìîé ìèøåíè êèíàçû ÌK-2 (ðèñ. 2; 3, á), à òàêæå ôîñôîðèëèðîâàíèå îäíîé èçîôîðìû JNK — áåëêà ð46 (ðèñ. 2). Òàêèì îáðàçîì, ñóïðåññèÿ ïèôèòðèíîì ð53-çàâèñèìîãî ïóòè ïðèâîäèëà ê ïðîäîëæèòåëüíîé àêòèâàöèè Âçàèìíîå âëèÿíèå p53/p21/Rb è MAP-êèíàçíûõ ñèãíàëüíûõ ïóòåé ERK1/2, p38 è JNK. Îäíàêî íåëüçÿ èñêëþ÷àòü âîçìîæíîñòü òîãî, ÷òî ïèôèòðèí ìîæåò çàïóñêàòü àêòèâàöèþ ÌÅK/ERK-êàñêàäà ÷åðåç ð53-íåçàâèñèìûé ìåõàíèçì (Kim et al., 2010). Ñóùåñòâóåò òàêæå èíòðèãóþùàÿ âîçìîæíîñòü àêòèâàöèè Raf/MEK/ERK-êàñêàäà ïîñðåäñòâîì Rb, ÷òî áûëî ïîêàçàíî íà ïðèìåðå ð53-ìóòàíòíûõ êëåòîê (Drosten et al., 2014). Îñíîâûâàÿñü íà ïðåäñòàâëåííûõ ðåçóëüòàòàõ, ìû ïðåäïîëàãàåì, ÷òî â ñòàðåþùèõ ÑÊÝ ôóíêöèîíàëüíî àêòèâíûé áåëîê ð53 íåãàòèâíî ðåãóëèðóåò àêòèâíîñòü ÌÀÐ-êèíàçíûõ êàñêàäîâ, ÷òî â èòîãå ìîæåò áûòü íåîáõîäèìî äëÿ ðàçâèòèÿ êëåòî÷íîãî ñòàðåíèÿ. Äåéñòâèå èíãèáèòîðîâ ÌÀÐ-êèíàçíûõ ê à ñ ê à ä î â. Ïðè èçó÷åíèè âëèÿíèÿ èíãèáèðîâàíèÿ ERK1/2, p38 èëè JNK íà ð53/ð21/Rb-ñèãíàëèíã â ÑÊÝ îêàçàëîñü, ÷òî ñóïðåññèÿ àêòèâíîñòè íè îäíîé èç ÌÀÐ-êèíàç â òå÷åíèå 30 ìèí íå ïðèâîäèëà ê èçìåíåíèþ óðîâíÿ Í2Î2-èíäóöèðîâàííîãî ôîñôîðèëèðîâàíèÿ ð53 (ðèñ. 1). Íàïðîòèâ, â óñëîâèÿõ èíãèáèðîâàíèÿ ÌÀÐ-êèíàçíûõ ïóòåé ÷åðåç 3 ñóò ôîñôîðèëèðîâàíèå ð53 çíà÷èòåëüíî óâåëè÷èâàëîñü ïî ñðàâíåíèþ ñ Í2Î2-ñòèìóëèðîâàííûìè êëåòêàìè (ðèñ. 2), ÷òî êîððåëèðîâàëî ñî çíà÷èòåëüíûì ïîâûøåíèåì ýêñïðåññèè áåëêà ð21 è ïîñëåäóþùèì ãèïîôîñôîðèëèðîâàíèåì áåëêà Rb (ðèñ. 3, à). Åñëè â ïðèñóòñòâèè ïèôèòðèíà áûëî âûÿâëåíî îæèäàåìîå ïîäàâëåíèå ýêñïðåññèè ð21, òî óñèëåíèå ýêñïðåññèè ð21 ïðè áëîêèðîâàíèè àêòèâíîñòè ÌÀÐ-êèíàç îêàçàëîñü äîâîëüíî íåîæèäàííûì ôàêòîì, êîòîðûé íàâîäèò íà ìûñëü î âîâëå÷åííîñòè ERK1/2, JNK è ð38 â ðåãóëÿöèþ ýêñïðåññèè ð21 íåçàâèñèìî îò àêòèâàöèè ð53. ×òî êàñàåòñÿ Rb, èíãèáèðîâàíèå êàæäîé èç ÌÀÐ-êèíàç âíîñèò äîïîëíèòåëüíûé âêëàä â íàáëþäàåìóþ ïîëíóþ äåçàêòèâàöèþ ýòîãî áåëêà íàðÿäó ñ èíãèáèðóþùèì ñèãíàëîì, ïåðåäàþùèìñÿ ÷åðåç ð53/ð21-ïóòü. Íà îñíîâå àíàëèçà âçàèìîâëèÿíèÿ ÌÀÐ-êèíàçíûõ è ð53/ð21/Rb-ñèãíàëüíûõ ïóòåé è ïðèíèìàÿ âî âíèìàíèå ïðîäîëæèòåëüíóþ àêòèâàöèþ ERK1/2, p38 èëè JNK ïðè èíãèáèðîâàíèè ð53 è, íàîáîðîò, àêòèâàöèþ ð53-îïîñðåäîâàííîãî ïóòè â îòâåò íà áëîêèðîâàíèå ëþáîé èç ÌÀÐ-êèíàç, ìû çàêëþ÷àåì, ÷òî èññëåäóåìûå ñèãíàëüíûå ïóòè â ÑÊÝ ôóíêöèîíèðóþò ïî ïðèíöèïó âçàèìíîãî ñóïðåññèðîâàíèÿ.  êëåòêàõ ñ ôóíêöèîíàëüíî íåàêòèâíûì áåëêîì ð53 êàê íà êîðîòêèõ ñðîêàõ ñòèìóëÿöèè Í2Î2, òàê è ÷åðåç íåñêîëüêî ñóòîê èç âñåõ ÌÀÐ-êèíàç èìåííî àêòèâàöèÿ ERK1/2 áûëà íàèáîëåå ñèëüíî âûðàæåííîé. Èñõîäÿ èç ýòèõ íàáëþäåíèé ìîæíî ïðåäïîëîæèòü, ÷òî â ïðîöåññå èíèöèàöèè è ðàçâèòèÿ ñòàðåíèÿ ïðè îêèñëèòåëüíîì ñòðåññå àêòèâèðîâàííûé áåëîê ð53 íåãàòèâíî ðåãóëèðóåò ERK1/2 (è â ìåíüøåé ñòåïåíè p38 èëè JNK). Ñîïîñòàâëåíèå ïîëó÷åííûõ íàìè ðåçóëüòàòîâ ñ ëèòåðàòóðíûìè äàííûìè ïîêàçûâàåò, ÷òî îíè â ïðèíöèïå ñîâïàäàþò ñ ðàáîòàìè, â êîòîðûõ óòâåðæäàåòñÿ ðîëü àêòèâèðîâàííîãî òðàíñêðèïöèîííîãî ôàêòîðà ð53 â êà÷åñòâå íåãàòèâíîãî ðåãóëÿòîðà àêòèâíîñòè ERK1/2 ÷åðåç ìåõàíèçì, âêëþ÷àþùèé ð53-çàâèñèìóþ èíäóêöèþ ñïåöèôè÷åñêèõ ôîñôàòàç (Bermudez et al., 2011; Zhang et al., 2015). Ñîãëàñíî ïîñëåäíèì ñîîáùåíèÿì, öèòîïëàçìàòè÷åñêàÿ ôîñôàòàçà ÌKÐ-3, ñïåöèôè÷íî äåôîñôîðèëèðóþùàÿ ERK1/2, âîâëå÷åíà â óñòàíîâëåíèå ôåíîòèïà, õàðàêòåðíîãî äëÿ ñòàðûõ êëåòîê (Zhang et al., 2015). Ìû àíàëèçèðîâàëè òàêæå âçàèìîäåéñòâèå ìåæäó ERK1/2, p38 è JNK, ïîî÷åðåäíî ïîäàâëÿÿ àêòèâíîñòü êàæäîé èç ýòèõ êèíàç ïîñðåäñòâîì ñîîòâåòñòâóþùèõ ôàðìàêîëîãè÷åñêèõ èíãèáèòîðîâ è îöåíèâàÿ óðîâåíü ôîñôîðèëèðîâàíèÿ ÌÀÐ-êèíàç. Èç íàèáîëåå èíòåðåñíûõ è çíà÷èìûõ ýôôåêòîâ ñëåäóåò îòìåòèòü ïðîòèâîïîëîæ- 793 Ðèñ. 4. Ñõåìà, îòðàæàþùàÿ âçàèìíîå âëèÿíèå ñèãíàëüíûõ ïóòåé â ÑÊÝ â óñëîâèÿõ îêèñëèòåëüíîãî ñòðåññà. MAP-êèíàçíûå êàñêàäû âêëþ÷àþò â ñåáÿ ERK, p38, MK-2 è JNK; èíãèáèòîð öèêëèíçàâèñèìûõ êèíàç p 21 è áåëîê ðåòèíîáëàñòîìû Rb ÿâëÿþòñÿ êîìïîíåíòàìè p53-îïîñðåäîâàííîãî ïóòè. íîå âëèÿíèå èíãèáèðîâàíèÿ p38 è JNK íà àêòèâàöèþ ERK. Äåéñòâèå SB203580 èíèöèèðîâàëî çàìåòíîå ïîâûøåíèå ôîñôîðèëèðîâàíèÿ ERK1/2, òîãäà êàê äåéñòâèå SP600125 ïðèâîäèëî ïðàêòè÷åñêè ê ïîëíîé äåçàêòèâàöèè ERK1/2 ÷åðåç 30 ìèí (ðèñ. 1) è çíà÷èòåëüíîìó óìåíüøåíèþ óðîâíÿ ôîñôîðèëèðîâàíèÿ ÷åðåç 3 ñóò (ðèñ. 2). Ñëåäîâàòåëüíî, ð38 ñóïðåññèðóåò àêòèâàöèþ ERK1/2 è äåéñòâóåò â ýòîì îòíîøåíèè ñèíåðãè÷íî ñ ð53, èíãèáèðóþùèé ýôôåêò êîòîðîãî îòìå÷åí âûøå. Íàïðîòèâ, ôóíêöèîíèðîâàíèå JNK ñâÿçàíî ñ ïîçèòèâíîé ðåãóëÿöèåé àêòèâíîñòè ERK1/2.  ñâîþ î÷åðåäü êèíàçû ERK1/2 ìîãóò àêòèâèðîâàòü JNK è ð38, ïîñêîëüêó ñëåäñòâèåì èíãèáèðîâàíèÿ ERK1/2 ÿâëÿåòñÿ ïàäåíèå óðîâíÿ ôîñôîðèëèðîâàíèÿ ð38 (ðèñ. 2), ÌK-2 (ðèñ. 3, á) è JNK (ðèñ. 1, 2) äî çíà÷åíèÿ íèæå êîíòðîëüíîãî. Åñëè â ñëó÷àå ð38 ïîíèæåíèå àêòèâàöèè ìîãëî áûòü ñëåäñòâèåì ïîíèæåíèÿ ýêñïðåññèè áåëêà ÷åðåç 3 ñóò, òî â ñëó÷àå JNK íàáëþäàëè èíâåðñèþ ýêñïðåññèè ð54 è ð46 èçîôîðì JNK ïî ñðàâíåíèþ ñ Í2Î2-îáðàáîòàííûìè êëåòêàìè, ÷òî âðÿä ëè ìîæåò áûòü ïðè÷èíîé äåçàêòèâàöèè JNK. Ðåçóëüòàòû ïðîâåäåííîãî èññëåäîâàíèÿ âçàèìîäåéñòâèÿ ñèãíàëüíûõ ïóòåé â òêàíåñïåöèôè÷íûõ ìåçåíõèìíûõ ÑÊÝ ÷åëîâåêà â óñëîâèÿõ îêèñëèòåëüíîãî ñòðåññà ïðåäñòàâëåíû â âèäå äèàãðàììû (ðèñ. 4), êîòîðàÿ äåìîíñòðèðóåò ñåòü ñëîæíûõ èíãèáèðóþùèõ è àêòèâèðóþùèõ ñâÿçåé ìåæäó ñîñòàâëÿþùèìè åå áåëêàìè. Ïîëó÷åííûå íîâûå äàííûå âíîñÿò âêëàä â ïîíèìàíèå ðåãóëÿòîðíûõ Ï. È. Äåðÿáèí è äð. 794 ìåõàíèçìîâ èíäóöèðîâàííîãî ñòðåññîì ñòàðåíèÿ ÑÊÝ è ìîãóò áûòü ïîëåçíû ïðè ðàçðàáîòêå áîëåå ýôôåêòèâíûõ ïîäõîäîâ, íàïðàâëåííûõ íà ïðåäîòâðàùåíèå ïðåæäåâðåìåííîãî ñòàðåíèÿ ñòâîëîâûõ êëåòîê ÷åëîâåêà ìåçåíõèìíîé ïðèðîäû. Ðàáîòà âûïîëíåíà ïðè ôèíàíñîâîé ïîääåðæêå Ðîññèéñêîãî ôîíäà ôóíäàìåíòàëüíûõ èññëåäîâàíèé (ïðîåêò 14-04-01720-à), Ðîññèéñêîãî íàó÷íîãî ôîíäà (ïðîåêò 14-50-00068) è ÔÀÍÎ Ðîññèè. Ñïèñîê ëèòåðàòóðû Çåìåëüêî Â. È., Ãðèí÷óê Ò. Ì., Äîìíèíà À. Ï., Àðöûáàøåâà È. Â., Çåíèí Â. Â., Êèðñàíîâ À. À., Áè÷åâàÿ Í. Ê., Êîðñàê Â. Ñ., Íèêîëüñêèé Í. Í. 2011. Ìóëüòèïîòåíòíûå ìåçåíõèìíûå ñòâîëîâûå êëåòêè äåñêâàìèðîâàííîãî ýíäîìåòðèÿ. Âûäåëåíèå, õàðàêòåðèñòèêà è èñïîëüçîâàíèå â êà÷åñòâå ôèäåðíîãî ñëîÿ äëÿ êóëüòèâèðîâàíèÿ ýìáðèîíàëüíûõ ñòâîëîâûõ ëèíèé ÷åëîâåêà. Öèòîëîãèÿ. 53 (12) : 919—929. (Zemel’ko V. I., Grinchuk T. M., Domnina A. P., Artzibasheva I. V., Zenin V. V., Kirsanov A. A., Bichevaya N. K., Korsak V. S., Nikolsky N. N. 2011. Multipotent mesenchymal stem cells of desquamated endometrium. Isolation, characterization, and application as a feeder layer for maintenance of human embryonic stem cells. Tsitologiya. 53 (12) : 919—929.) Bennett B. L., Sasaki D. T., Murray B. W., O’Leary E. C., Sakata S. T., Xu W., Leisten J. C., Motiwala A., Pierce S., Satoh Y., Bhagwat S. S., Manning A. M., Anderson D. W. 2001. SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc. Nat. Acad. Sci. USA. 98 : 13 681—13 686. Bermudez O., Jouandin P., Rottier J., Bourcier C., Pages G., Gimond C. 2011. Post-transcriptional regulation of the DUSP6 /MKP-3 phosphatase by MEK/ERK signaling and hypoxia. J. Cell. Physiol. 226 : 276—284. Bogoyevitch M. A., Kobe B. 2006. Uses for JNK: the many and varied substrates of the c-Jun N-terminal kinases. Microbiol. Mol. Biol. Rev. 70 : 1061—1095. Borodkina A., Shatrova A., Abushik P., Nikolsky N., Burova E. 2014. Interaction between ROS dependent DNA damage, mitochondria and p38 MAPK underlies senescence of human adult stem cells. Aging. 6 : 481—495. Burova E., Borodkina A., Shatrova A., Nikolsky N. 2013. Sublethal oxidative stress induces the premature senescence of human mesenchymal stem cells derived from endometrium. Oxid. Med. Cell. Longev. 2013 : 474931. Burova E., Vassilenko K., Dorosh V., Gonchar I., Nikolsky N. 2007. Interferon gamma-dependent transactivation of epidermal growth factor receptor. FEBS Lett. 581 : 1475—1480. Chang L., Karin M. 2001. Mammalian MAP kinase signalling cascades. Nature. 410 : 37—40. Debacq-Chainiaux F., Boilan E., Dedessus Le Moutier J., Weemaels G., Toussaint O. 2010. p38 (MAPK) in the senescence of human and murine fibroblasts. Adv. Exp. Med. Biol. 694 : 126— 137. Drosten M., Sum E. Y., Lechuga C. G., Simón-Carrasco L., Jacob H. K., Garcia-Medina R., Huang S., Beijersbergen R. L., Bernards R., Barbacid M. 2014. Loss of p53 induces cell proliferation via Ras-independent activation of the Raf/Mek/Erk signaling pathway. Proc. Nat. Acad. Sci. USA. 111 : 15 155—15 160. Duan W. J., Li Q. S., Xia M. Y., Tashiro S., Onodera S., Ikejima T. 2011. Silibinin activated p53 and induced autophagic death in human fibrosarcoma HT1080 cells via reactive oxygen species-p38 and c-Jun N-terminal kinase pathways. Biol. Pharm. Bull. 34 : 47—53. Fang L., Li G., Liu G., Lee S. W., Aaronson S. A. 2001. p53 induction of heparin-binding EGF-like growth factor counteracts p53 growth suppression through activation of MAPK and PI3K/Akt signaling cascades. EMBO J. 20 : 1931—1939. Favata M. F., Horiuchi K. Y., Manos E. J., Daulerio A. J., Stradley D. A., Feeser W. S., Van Dyk D. E., Pitts W. J., Earl R. A., Hobbs F., Copeland R. A., Magolda R. L., Scherle P. A., Trzaskos J. M. 1998. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J. Biol. Chem. 273 : 18 623— 18 632. Fujisawa T., Takeda K., Ichijo H. 2007. ASK family proteins in stress response and disease. Mol. Biotechnol. 37 : 13—18. Guan Q. H., Pei D. S., Zong Y. Y., Xu T. L., Zhang G. Y. 2006. Neuroprotection against ischemic brain injury by a small peptide inhibitor of c-Jun N-terminal kinase (JNK) via nuclear and non-nuclear pathways. Neuroscience. 139 : 609—627. Gudkov A. V., Komarova E. A. 2005. Prospective therapeutic applications of p53 inhibitors. Biochem. Biophys. Res. Commun. 331 : 726—736. Hsu S. P., Ho P. Y., Liang Y. C., Ho Y. S., Lee W. S. 2009. Involvement of the JNK activation in terbinafine-induced p21 up-regulation and DNA synthesis inhibition in human vascular endothelial cells. J. Cell. Biochem. 108 : 860—866. Kaji A., Zhang Y., Nomura M., Bode A. M., Ma W. Y., She Q. B., Dong Z. 2003. Pifithrin-alpha promotes p53-mediated apoptosis in JB6 cells. Mol. Carcinog. 37 : 138—148. Kim E. K., Choi E. J. 2015. Compromised MAPK signaling in human diseases: an update. Arch. Toxicol. 89 : 867—882. Kim S., Han J., Lee S. K., Hur S. M., Koo M., Cho D. H., Bae S. Y., Choi M. Y., Shin I., Yang J. H., Nam S. J., Lee J. E. 2010. Pifithrin-alfa, an Inhibitor of p53 transactivation, up-regulates COX-2 expression through an MAPK-dependent pathway. Pharmacology. 86 : 313—319. Komarov P. G., Komarova E. A., Kondratov R. V., ChristovTselkov K., Coon J. S., Chernov M. V., Gudkov A. V. 1999. A chemical inhibitor of p53 that protects mice from the side effects of cancer therapy. Science. 285 : 1733—1737. Kyriakis J. M., Avruch J. 2012. Mammalian MAPK signal transduction pathways activated by stress and inflammation: a 10-year update. Physiol. Rev. 92 : 689—737. Lee S. W., Fang L., Igarashi M., Ouchi T., Lu K. P., Aaronson S. A. 2000. Sustained activation of Ras/Raf/mitogen-activated protein kinase cascade by the tumor suppressor p53. Proc. Nat. Acad. Sci. USA. 97 : 8302—8305. Lee S. Y., Shin S. J., Kim H. S. 2013. ERK1/2 activation mediated by the nutlin—3-induced mitochondrial translocation of p53. Int. J. Oncol. 42 : 1027—1035. Lewis T. S., Shapiro P. S., Ahn N. G. 1998. Signal transduction through MAP kinase cascades. Adv. Cancer Res. 74 : 49—139. Lin T., Mak N. K., Yang M. S. 2008. MAPK regulate p53-dependent cell death induced by benzo[a]pyrene: involvement of p53 phosphorylation and acetylation. Toxicology. 247 : 145—153. Lorin S., Pierron G., Ryan K. M., Codogno P., Djavaheri-Mergny M. 2010. Evidence for the interplay between JNK and p53DRAM signalling pathways in the regulation of autophagy. Autophagy. 6 : 153—154. McCubrey J. A., Steelman L. S., Chappell W. H., Abrams S. L., Wong E. W., Chang F., Lehmann B., Terrian D. M., Milella M., Tafuri A., Stivala F., Libra M., Basecke J., Evangelisti C., Martelli A. M., Franklin R. A. 2007. Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Biochim. biophys. acta. 1773 : 1263—1284. Menendez D., Inga A., Resnick M. A. 2009. The expanding universe of p53 targets. Nat. Rev. Cancer. 9 : 724—737. Ongusaha P. P., Kim J. I., Fang L., Wong T. W., Yancopoulos G. D., Aaronson S. A., Lee S. W. 2003. p53 induction and activation of DDR1 kinase counteract p53-mediated apoptosis and influence p53 regulation through a positive feedback loop. EMBO J. 22 : 1289—1301. Passos J. F., Nelson G., Wang C., Richter T., Simillion C., Proctor C. J., Miwa S., Olijslagers S., Hallinan J., Wipat A., Saretzki G., Rudolph K. L., Kirkwood T. B., von Zglinicki T. 2010. Feedback between p21 and reactive oxygen production is necessary for cell senescence. Mol. Syst. Biol. 6 : 347. Pearson G., Robinson F., Beers Gibson T., Xu B. E., Karandikar M., Berman K., Cobb M. H. 2001. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr. Rev. 22 : 153—183. Âçàèìíîå âëèÿíèå p53/p21/Rb è MAP-êèíàçíûõ ñèãíàëüíûõ ïóòåé Persons D. L., Yazlovitskaya E. M., Pelling J. C. 2000. Effect of extracellular signal-regulated kinase on p53 accumulation in response to cisplatin. J. Biol. Chem. 275 : 35 778—35 785. Porras A., Zuluaga S., Black E., Valladares A., Alvarez A. M., Ambrosino C., Benito M., Nebreda A. R. 2004. P38 alpha mitogen-activated protein kinase sensitizes cells to apoptosis induced by different stimuli. Mol. Biol. Cell. 15 : 922—933. Riley T., Sontag E., Chen P., Levine A. 2008. Transcriptional control of human p53-regulated genes. Nat. Rev. Mol. Cell. Biol. 9 : 402—412. Sawe N., Steinberg G., Zhao H. 2008. Dual roles of the MAPK/ERK1/2 cell signaling pathway after stroke. J. Neurosci. Res. 86 : 1659—1669. She Q. B., Bode A. M., Ma W. Y., Chen N. Y., Dong Z. 2001. Resveratrol-induced activation of p53 and apoptosis is mediated by extracellular-signal-regulated protein kinases and p38 kinase. Cancer Res. 61 : 1604—1610. Singh S., Upadhyay A. K., Ajay A. K., Bhat M. K. 2007. p53 regulates ERK activation in carboplatin induced apoptosis in cervical carcinoma: a novel target of p53 in apoptosis. FEBS Lett. 581 : 289—295. Takeda K., Naguro I., Nishitoh H., Matsuzawa A., Ichijo H. 2011. Apoptosis signaling kinases: from stress response to health outcomes. Antioxid. Redox. Signal. 15 : 719—761. 795 Wu G. S. 2004. The functional interactions between the p53 and MAPK signaling pathways. Cancer Biol. Ther. 3 : 156— 161. Xia Z., Dickens M., Raingeaud J., Davis R. J., Greenberg M. E. 1995. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science. 270 : 1326—1331. Xiao Y., Yan W., Lu L., Wang Y., Lu W., Cao Y., Cai W. 2015. p38/p53/miR-200a-3p feedback loop promotes oxidative stressmediated liver cell death. Cell Cycle. 14 : 1548—1558. Young P. R., McLaughlin M. M., Kumar S., Kassis S., Doyle M. L., McNulty D., Gallagher T. F., Fisher S., McDonnell P. C., Carr S. A., Huddleston M. J., Seibel G., Porter T. G., Livi G. P., Adams J. L., Lee J. C. 1997. Pyridinyl imidazole inhibitors of p38 mitogen?activated protein kinase bind in the ATP site. J. Biol. Chem. 272 : 12 116—12 121. Zarubin T., Han J. 2005. Activation and signaling of the p38 MAP kinase pathway. Cell Res. 15 : 11—18. Zhang H., Chi Y., Gao K., Zhang X., Yao J. 2015. p53 protein-mediated up-regulation of MAP kinase phosphatase 3 (MKP-3) contributes to the establishment of the cellular senescent phenotype through dephosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). J. Biol. Chem. 290 : 1129—1140. Ïîñòóïèëà 27 VIII 2015 RELATIONSHIP BETWEEN p53/p21/Rb AND MAPK SIGNALING PATHWAYS IN HUMAN ENDOMETRIUM-DERIVED STEM CELLS UNDER OXIDATIVE STRESS P. I. Deryabin, A. V. Borodkina, N. N. Nikolsky, E. B. Burova1 Institute of Cytology RAS, St. Petersburg, 194064; 1 e-mail: [email protected] Human endometrium-derived mesenchymal stem cells (hMESC) under the sublethal oxidative stress induced by H2O2 activate both p53/p21/Rb and p38MAPK/MAPKAPK-2 pathways that are responsible for the induction of hMESC premature senescence (Borodkina et al., 2014). However the mutual relations between p53/p21/Rb and MAPK signaling pathways, including ERK, p38 and JNK remain unexplored as yet. Here, we used the specific inhibitors — pifithrin-a (PFT), U0126, SB203580 and SP600125 to «switch off» one of the proteins in these cascades and to evaluate the functional status alterations of the rest proteins. Suppression each of the MAPK significantly increased the p53 phosphorylation levels, as well as p21 protein expression followed by Rb hypophosphorylation. On the other hand, PFT-induced p53 inhibition enhanced mostly the ERK1/2 activation compared with p38 and JNK. These results suppose the existence of the reciprocal negative regulation between p53- and MAPK-dependent signaling pathways. Analyzing the possible interactions among the members of the MAPK family, we showed that p38 and JNK can function as the ERK antagonists: JNK is capable to activate ERK, while p38 may block the ERK activation. Together, these results demonstrate complex links between different signaling cascades in stressed hMESC, implicating ERK, p38 and JNK in regulation of the premature senescence via p53/p21/Rb pathway. K e y w o r d s: premature senescence, MAPK, pifithrin-a, p53, endometrial stem cells, oxidative stress, signaling pathways.
