The association between tissue non-specific alkaline phosphatase expression and differentiation of mesenchymal stromal cells

Bartlett, Cara-Lesley (2017-03)

Thesis (D.Phil)--Stellenbosch University, 2017.

Thesis

ENGLISH SUMMARY: Diseases resulting from the dysregulation of adipocyte and osteoblast differentiation include diabetes type II and osteoporosis. Both adipocytes and osteoblasts are derived from the same progenitor cell type, known as mesenchymal stromal cells (MSCs), which may also differentiate into cells of several mesenchymal lineages. A more detailed understanding of the mechanisms involved in the differentiation of MSCs would provide valuable insight into the underlying causes of, as well as facilitate the development of improved treatments for, diseases related to MSC differentiation dysregulation. Tissue-nonspecific alkaline phosphatase (TNAP) is highly expressed in several tissues including bone tissue, where it has a well-established role in skeletal mineralisation. In recent years TNAP expression has been reported in adipocytes, and has been identified as identical to the stem cell marker, mesenchymal stem cell antigen-1 (MSCA-1). The above findings indicate that TNAP has diverse roles, and may be one of the factors involved in determining the differentiation pathway of MSCs. Previous studies have found that inhibition of TNAP in the mouse preadipocyte cell line, 3T3-L1 resulted in a decrease in lipid accumulation during in vitro adipogenic differentiation, suggesting that TNAP is involved in adipogenesis. In the present study, rat-derived primary MSCs were isolated from bone marrow (bmMSCs) as well as subcutaneous (scADSCs) and peri-renal visceral adipose (pvADSCs) depots, and differentiated in vitro towards either an adipocytic or osteoblastic phenotype. The expression of TNAP was assessed in rat-derived MSCs undergoing both adipogenic and osteogenic differentiation. TNAP expression levels were highest in bmMSCs, followed by scADSCs and pvADSCs, with higher alkaline phosphatase (ALP) activity observed during adipogenesis compared to osteogenesis in all three MSC types. The addition of the reported TNAP inhibitor, levamisole during osteogenesis prevented mineralisation in all MSC types, but had no significant effect on lipid accumulation during adipogenesis. Other reported inhibitors were also examined; Histidine was not successful in reducing lipid accumulation or mineralisation, while Lhomoarginine was able to significantly reduce lipid accumulation in all MSC types. The inhibitor results were not conclusive due to possible off target effects within the cells. Attempts to inhibit adipogenic differentiation by knockdown of TNAP expression in scADSCs using shRNA were not successful, as indicated by the presence of lipid droplets in cells where TNAP-specific shRNA was present. This study also revealed that ALP activity was localised to the membrane of intracellular lipid droplets characteristic of adipocytes, and that the same TNAP mRNA transcript type which is preferentially expressed in bone tissue is also preferably expressed during adipogenic differentiation of bmMSCs and scADSCs, while expression in pvADSCs was below detectable levels. TNAP isoforms differ from one another due to differences in posttranslational glycosylation pattern. Glycosylation differences were observed between bmMSCs differentiated from a naïve state towards an adipogenic, compared to an osteogenic, phenotype. Differences were also observed between scADSCs and bmMSCs when differentiated towards adipocytes. This may indicate that a distinct isoform of TNAP exists in adipocytes. In conclusion, this study confirms earlier findings on the presence of TNAP in adipocytes. Differences in TNAP expression from MSCs isolated from different tissue depots were also discovered. This study provides a characterisation of the role of TNAP in adipogenic differentiation; however, the exact mechanisms remain to be elucidated.

AFRIKAANS OPSOMMING: Die wanregulering van adiposiet en osteoblast differensiasie lei tot verskeie siektetoestande, insluitende tipe II diabetes en osteoporosis. Beide adiposiet en osteoblast seltipes is afstammelinge van dieselfde stamseltipe, naamlik mesenchiem stromale selle (MSSe). Meer breedvoerige kennis van die onderliggende faktore wat bydra tot hierdie wanregulering kan aanleiding gee tot verbeterde behandelingsmetodes vir siektes geassosieer met afwykende MSS differensiasie. Daar is ‘n hoë uitdrukking van weefsel nie-spesifieke alkaliese fosfatase (WNAF) in verskeie weefseltipes, insluitende been, waar dit ‘n welbekende rol speel in skelet mineralisering het. Daar is onlangs bevind dat WNAF in adiposiete uitgedruk word en dat dit identies is tot die stamsel merker, mesenchiem stamsel antigeen-1. Hierdie bevindinge dui daarop dat WNAF verskeie diverse funksies het en dat die ensiem een van die bepalende faktore mag wees wat bydra tot differensiasie van stamselle. Vorige navorsing het gewys dat inhibisie van WNAF in die muis pre-adiposiet sellyn, 3T3L1, lei tot ‘n afname in lipied akkumulasie tydens in vitro adipogeniese differensiasie. Dit impliseer dat WNAF betrokke is by adipogenese. In die huidige studie was daar gebruik gemaak van rot-afgeleide primêre MSSe geïsoleer uit beenmurg (bmMSSe), asook selle verkry uit subkutaan (skAASSe) en peri-renale visserale (pvAASSe) vet depots. Hierdie selle was gedifferensieer, in vitro, in ‘n adiposiet of osteoblast fenotipe. Die uitdrukking van WNAF was bepaal in rot-afgeleide MSSe wat gestimuleer was om of adipogeniese of osteogeniese differensiasie te ondergaan. Vlakke van WNAF uitdrukking was die hoogste in bmMSSe gevolg deur skAASSe en pvAASSe. Daar was meer alkaliese fosfatase aktiwiteit tydens adipogenese in al drie MSS tipes. Verder het die WNAF inhibitor, levamisool, mineralisering tydens osteogenese voorkom in al drie MSS tipes. Die inhibitor het egter geen beduidende effek gehad op lipied akkumulasie tydens adipogenese nie. Die effek van twee ander moontlike WNAF inhibitore, histidien en L-homoarginien, was ook ondersoek. Histidien het nie lipied akkumulasie of mineralisering onderdruk nie. Inteenstelling hiermee het L-homoarginien lipied akkumulasie beduidend in al drie MSS tipes verminder. Resultate verkry met inhibitore was nie beslissend nie. Dit mag wees as gevolg van moontlike buite-teiken effekte van die inhibitore in hierdie selle. Dus was daar gepoog om adiposiet differensiasie te inhibeer deur die afklop van WNAF uitdrukking in skAASSe. Dit was bewerkstellig deur gebruik te maak van klein haarnaald RNAs (khRNAs). Selle het egter steeds lipied druppels vertoon in die teenwoordigheid van WNAF spesifieke khRNAs. Eksperimente in hierdie tesis het ook onthul dat WNAF aktiwiteit gelokaliseer is in die membraan van intrasellulêre lipied druppels. Verder is daar ook gedemonstreer dat dieselfde WNAF boodskapper RNA-transkripsie tipe wat met voorkeur in beenweefsel uitgedruk word, ook met voorkeur tydens adipogeniese differensiasie uitgedruk word in bmMMSe en skAASSe. Die uitdrukking van hierdie transkripsie tipe was nie in pvAASSe gevind nie. Verskille in post-translasie glikosilasie gee aanleiding to verkillende isoforme van WNAF. Glikosilasie-verskille was gesien in MSSe differensiasie in ‘n adipogeniese fenotipe in vergelyking met differensiasie in ‘n osteogeniese fenotipe. So ook was daar verkille gemerk tussen skAASSe en bmMSSe wat gedifferensieer was in ‘n adiposiet fenotipe. Dit mag aandui dat ‘n eiesoortige WNAF isoform in adiposiete voorkom. Bogenoemde saamgevat, die huidige studie bevestig vroeëre bevindinge dat WNAF teenwoordig is in adiposiete. Verskille is gewys in die uitdrukking van WNAF in MSSe geïsoleer uit versillende weefsel depots. Hierdie studie voorsien ‘n karakterisering van die rol wat WNAF speel in adipogeniese differensiasie, maar die presiese funksie van WNAF is egter nog ontwykend.

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