Examining plasminogen activator-inhibitor-1 and C-reactive protein regulation by glucocorticoids and pro-inflammatory cytokines in liver cell lines

Dale, Lieke (2021-12)

Thesis (MSc)--Stellenbosch University, 2021.

Thesis

ENGLISH ABSTRACT: Current knowledge suggests that a continued elevated acute phase response (APR), which is often referred to as low-grade inflammation, occurs during insulin resistance. The levels of acute phase proteins (APPs), such as plasminogen activator-inhibitor-1 (PAI-1), C-reactive protein (CRP), and serum amyloid A (SAA) are significantly increased during this response, hence why they are commonly used as biological markers for type 2 diabetes (T2D). In the liver, APPs are regulated by the peripheral mediators of stress (i.e., endogenous glucocorticoids (GCs)) and inflammation (i.e., pro-inflammatory cytokines), with both implicated in the development of insulin resistance. Exogenous GCs are routinely prescribed in the management of inflammatory disorders. However, long-term GC treatment can exaggerate insulin resistance as it could possibly disrupt the interplay between endogenous GCs and cytokines, leading to a recurrent, chronic APR. Current knowledge suggests that when it comes to the APR, GCs, which are traditionally known for its anti-inflammatory properties, exert more pro- inflammatory behaviour, by further strengthening cytokine-induced APP expression. Whilst this cooperative regulation by GCs and pro-inflammatory cytokines have been extensively studied and demonstrated for SAA, fewer have studied this phenomenon in regards to PAI-1 and CRP, especially in the liver. In addition, previous studies investigating the possible co- regulation of PAI-1 and CRP by GCs and pro-inflammatory cytokines, included only interleukin-6 (IL-6) or tumour necrosis factor-alpha (TNF-α) as representative pro- inflammatory cytokine, respectively. Therefore, the current study examined the expression of both PAI-1 and CRP in response to GCs (synthetic and endogenous) in the presence of either pro-inflammatory cytokine, TNF-α or IL-6, in a murine (BWTG3)- and human (HepG2) liver cell line. The effects were investigated at both the mRNA- and intracellular protein level, using real-time semi-quantitative polymerase chain reaction (qPCR) and western blotting, respectively. In addition, the potential underlying molecular mechanism governing the action of these biological mediators to affect APP expression were investigated, using a luciferase promotor-reporter assay. PAI-1 and CRP levels generally remained elevated in response to individual and combinatorial treatments with GCs and pro-inflammatory cytokines, at intracellular protein- and promotor level. However, only under certain conditions, cooperativity was displayed between the test compounds, where PAI-1 and CRP expression were further potentiated, more than what was observed with either test compound alone. APPs are secreted proteins; therefore, the rate of protein synthesis and export presumably plays a role in determining intracellular APP expression and highlights the importance of measuring both intra- and extracellular APP expression, the latter of which the current study did not determine. In addition, the increase in PAI-1 and CRP promoter activity does suggest that the molecular mechanism of action whereby GCs and pro-inflammatory cytokines increase PAI-1 and CRP levels is at the promoter level. Whether only the proximal promotor is involved remains to be elucidated. Taken together, the findings of the current study give credence to the current knowledge available on the subject of cooperative regulation of APPs by GCs and pro- inflammatory cytokines. It is clear that GCs favour an increase in APP expression, at times enhancing the cytokine-induced PAI-1 and CRP expression. However, GCs were also able to antagonise the cytokine-induced PAI-1 and CRP expression, which is in line with their anti- inflammatory role. Ultimately, our findings highlight the diversity and complexity to the often- contradictory nature of GCs and their crosstalk with inflammatory mediators. Keywords: acute phase proteins, acute phase response, cooperative regulation, C-reactive protein, glucocorticoids, hepatic insulin resistance, inflammation, plasminogen activator- inhibitor-1, pro-inflammatory cytokines, stress

AFRIKAANSE OPSOMMING: Bestaande kennis dui daarop dat ’n verhoogde akute fase reaksie (AFR), ook bekend as lae- graadse inflammasie, tydens insulienweerstandigheid voorkom. Die vlakke van akute fase proteïene (AFPe), soos plasminogeen aktiveerder-inhibeerder-1 (PAI-1), C-reaktiewe proteïen (CRP) en serum amiloïed A (SAA) word aansienlik verhoog tydens hierdie reaksie, en word dus gereeld gebruik as biologiese merkers vir tipe 2 diabetes (T2D). In die lewer word AFPe deur die perifere molekules van stres (endogene glukokortikoïede (GKe)) en inflammasie (pro- inflammatoriese sitokiene) gereguleer, met beide wat ook in die ontwikkeling van insulienweerstandigheid geïmpliseer word. Eksogene GKe word gereeld voorgeskryf in die behandeling van inflammatoriese afwykings. Langtermyn GK-behandeling kan egter insulienweerstandigheid vererger, omdat dit dalk die wisselwerking tussen endogene GKe en sitokiene kan versteur, wat tot ’n herhaalde, chroniese AFR kan lei. Bestaande kennis dui daarop dat, met betrekking tot die AFR, GKe, wat tradisioneel bekend is vir hul anti- inflammatoriese eienskappe, geneig is om meer pro-inflammatories op te tree deur sitokien- geïnduseerde AFP-uitdrukking verder te versterk. Hoewel hierdie samewerkende regulering deur GKe en pro-inflammatoriese sitokiene vir SAA breedvoerig bestudeer en gedemonstreer is, is daar minder studies oor dié verskynsel ten opsigte van PAI-1 en CRP, veral in die lewer. Daarby het vorige studies ook slegs interleukin-6 (IL-6), in die geval van CRP, of tumor nekrose faktor-alfa (TNF-α), in die geval van PAI-1, gebruik as verteenwoordiger sitokien in kombinasie met die GK. Die huidige studie ondersoek dus die uitdrukking van beide PAI-1 en CRP in reaksie op GKe (sinteties en endogeen) in die teenwoordigheid van óf pro- inflammatoriese sitokien, TNF-α of IL-6, in ’n muriene (BWTG3) en menslike (HepG2) lewersellyn. Die uitwerkings is op beide mRNA- en intrasellulêre proteïenvlak ondersoek, met behulp van onderskeidelik intydse semi-kwantitatiewe polimerase kettingreaksie en western klad. Daarbenewens is die moontlike onderliggende molekulêre meganisme ondersoek, wat die aksie van hierdie biologiese molekules beheer om die uitdrukking van beide AKPe te beïnvloed, met behulp van ’n lusiferase promotor-verslaggewer toets. Die vlakke van PAI-1 en CRP was meestal verhoog in reaksie op individuele en gesamentlike behandelings met GKe en pro-inflammatoriese sitokiene, op proteïen- en promotorvlak. Maar samewerking tussen die toetsverbindings is slegs onder sekere omstandighede getoon, waar PAI-1- en CRP-uitdrukking verder versterk was, meer as wat met elke toetsverbinding op sy eie waargeneem was. AFPe is afgeskeide proteïene; daarom speel die tempo van proteïensintese en -uitvoer vermoedelik ’n rol om intrasellulêre AFP-uitdrukking te bepaal, en beklemtoon dit die belangrikheid om beide intra- en ekstrasellulêre AFP-uitdrukking te bepaal, waarvan laasgenoemde nie in die huidige studie uitgevoer is nie. Verder dui die toename in PAI-1- en CRP promotor-aktiwiteit daarop dat die molekulêre aksiemeganisme waardeur GKe en pro-inflammatoriese sitokiene die vlakke van PAI-1- en CRP verhoog, op die promotorvlak is. Of slegs die proksimale promotor betrokke is, moet nog bepaal word. Gesamentlik gee die bevindinge van die hierdie studie geloofwaardigheid aan die beskikbare kennis oor die onderwerp van samewerkende regulering van AFPe deur GKe en pro-inflammatoriese sitokiene. Dit is duidelik dat GKe ’n toename in AFP-uitdrukking verkies, wat onder sekere omstandighede die sitokien-geïnduseerde PAI-1- en CRP-uitdrukking verbeter. GKe kan ook egter die sitokien-geïnduseerde PAI-1- en CRP- uitdrukking teenstaan, wat in lyn is met hul anti-inflammatoriese rol. Ten slotte, beklemtoon ons bevindings die diversiteit en kompleksiteit van die dikwels teenstrydige aard van GKe en hul kruiskommunikasie met inflammatoriese molekules. Sleutelwoorde: akute fase-proteïene, akute fase reaksie, C-reaktiewe proteïen, glukokortikoïede, hepatiese insulienweerstandigheid, inflammasie, ko-regulering, plasminogeen aktiveerder-inhibeerder-1, pro-inflammatoriese sitokiene, stres

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