Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Insulin is a mitogen for many cells and commonly signals through the classical, mitogenic Raf-
MEK-ERK or metabolic PB-kinase pathways. Insulin deficiency or type I diabetes causes
severe osteopenia. Obese patients with type II diabetes or insulin resistance, a disease associated
with defective insulin signaling pathways and high levels of circulating insulin, have increased
or normal bone mineral density. The question of whether hyperinsul inemia preserves bone mass
is frequently raised. However, there is still a lot of controversy on the role of insulin as an
osteoanabolic agent and this question still remains unanswered. A critical role for insulin
signaling in bone building osteoblasts has recently been demonstrated with IRS-l knock-out
mice. These mice developed low-turnover osteopenia due to impaired proliferation and
differentiation, stressing the importance of osteoblastic IRS-l for maintaining normal bone
formation.
In the present study it was found that insulin does function in vitro as an osteoblast mitogen.
This was illustrated in three relatively immature osteoblast (MBA-15.4, -15.6 mouse and MG-
63 human) cell lines, which responded to insulin with significant increases in proliferation. In
the MBA -15.4 preosteoblasts insulin stimulation of proliferation was comparable to the welldescribed
mitogen, TPA. The UMR-I06 cell line expresses markers of differentiated
osteoblasts, and was much less responsive to insulin treatment. The difference in proliferative
potential may be due to differences between spontaneously transformed cell lines, or the stage
of cell differentiation.
UOI26, a MEKI/2 inhibitor and wortmannin, a PB-kinase inhibitor, were used to investigate the
pathway used by insulin to signal and activate ERK and osteoblast proliferation. In MBA-15.4
mouse preosteoblasts, GF-containing FCS was completely dependent on MEK for DNA
synthesis. In contrast, in both MBA-15.4 and more mature MBA-15.6 osteoblasts, insulininduced
proliferation was resistant to the inhibitors alone or in combination. Higher MEKinhibitor
concentrations had no effect, and proliferation was also increased by the inhibitors in
several experiments. This indicated that the classical, insulin mitogenic pathway was not
involved in MBA-15.4 proliferation. Wortmannin had no effect on either insulin- or 20% FCSstimulated
proliferation, but inhibited activation of Akt/PKB, the metabolic downstream target
of PI3-kinase. Insul in signal ing to ERK was both MEK-and PI3-kinase- dependent, but this had
no effect on proliferation. In contrast, FCS-stimulated ERK activation and proliferation was
almost completely dependent on MEK-ERK activation. Proliferative signaling in the MG-63 human osteoblastic cell line in response to insulin was
partially dependent on MEK and partially dependent on PB-kinase. In contrast, signaling in
response to the phorbol ester, TPA, was partially dependent on PI3K but totally dependent on
MEK-ERK. This indicates that the signal converges on ERK, suggesting the involvement of a
PB-kinase upstream of a dominant MEK-ERK pathway. The differences found here between
mouse and human insulin mitogenic signaling pathways indicate that there may be species
differences between osteoblast signaling pathways, with mouse cells being independent and
human cells being dependent on MEK for DNA synthesis in response to insulin.
The effects of glucocorticoids on insulin mitogenic signaling in osteoblasts were also
investigated, because chronic long-term steroid use results in excessive bone loss. The PTP
inhibitor, sodium orthovanadate, reversed GC-impaired TPA- and FCS- induced proliferation in
MBA-1SA and MG-63 preosteoblasts. PTPs, such as SHP-l and PTP-IB, dephosphorylate and
inactivate phosphorylated kinases. Both SHP-l and PTPlB associated with kinases in the
mitogenic signaling cascade of MBA-lS.4 preosteoblasts growing rapidly in 10% FCS. Further,
SHP-I co-irnmunoprecipitated with active, tyrosine phosphorylated ERK, which may indicate
that it can dephosphorylate and inactivate ERK. However, since the MEK-ERK or PB-kinase
pathways are not important in insulin-induced proliferation in mouse osteoblasts, the PTPs are
unlikely to be role players in the negative regulation of this signaling pathway. This was
confirmed by the finding that vanadate was unable to reverse GC-induced decreases in insulinstimulated
DNA synthesis. This suggests that vanadate-sensitive PTPs may not be important in
the negative regulation of insulin-induced mouse osteoblast proliferation, and provides further
evidence of a novel insulin mitogenic pathway in the MBA-lSA but not MG-63 osteoblastic
cell line. / AFRIKAANSE OPSOMMING: Insulien is 'n mitogeen vir baie selle en gelei na binding aan die insulien reseptor, intrasellulêre
seine via die klassieke, mitogeniese Raf-MEK-ERK of die metaboliese PB-kinase
seintransduksie pad. 'n Insulien gebrek of tipe I diabetes veroorsaak osteopenie. Vetsugtige
pasiënte met insulien weestandigheid of tipe II diabetes, 'n siekte wat geassosieer word met
foutiewe insulien seintransduksie en hoë vlakke van sirkuierende insulien, het verhoogde of
normale been mineraal digtheid (BMD). Die vraag of hiper insulin ernie 'n verlies aan beenmassa
teëwerk word dikwels gevra. Teenstrydigheid oor die rol van insulien as 'n osteo-anaboliese stof
bestaan egter steeds en hierdie vraag bly dus onbeantwoord. Dat insulien seintransduksie wel 'n
kritiese rol speel in beenvormende osteoblaste is onlangs bevestig in studies met muise waarvan
die geen vir IRS-l uitgeslaan is. Hierdie muise ontwikkel 'n lae omset osteopenie weens
verswakte proliferasie en differensiasie.
fn hierdie studie is gevind dat insulien wel in vitro as 'n osteoblast mitogeen kan funksioneer.
Dit is in drie relatief onvolwasse (MBA-15.4, -15.6 muis en MG-63 mens) sellyne geillistreer,
deur betekenisvolle verhogings in insulien-geaktiveerde proliferasie. In MBA-15.4 preosteoblaste
is die persentasie verhoging in insulien-gestimuleerde proliferasie vergelykbaar met
dié van die bekende mitogeniese forbolester, TPA. Die UMR-I06 sellyn het kenmerke van
gedifferensieerde osteoblaste, en was baie minder responsief op insulien behandeling. Die
verskil in die proliferasie vermoë van die verskillende sellyne kan die gevolg wees van verskille
wat bestaan tussen spontaan getransformeerde sellyne of die stadium van sel differensiasie.
'n MEK 1/2 inhibitor, UO126 en 'n PB-kinase inhibitor, wortmannin, is gebruik om die insulien
seintransduksie pad noodsaaklik vir die aktivering van ERK en osteoblast proliferasie te bepaal.
In MBA-1S.4 muis pre-osteoblaste, was fetale kalf SenlTI1(FKS)-geinduseerde DNA sintese
totaal afhanklik van MEK. Beide die MBA-15.4 en die meer volwasse MBA-15.6 muis
osteoblaste was weerstandig teen die inhibitors op hulle eie, of in kombinasie. Verhoogde
MEK-inhibitor konsentrasies het geen verdere effek gehad nie en in verskeie eksperimente is 'n
verhoging in preliferasie selfs waargeneem met MEK-inhibisie. Hierdie resultate dui aan dat die
klassieke insulien mitogeniese pad nie betrokke is in MBA-I5.4 gestimuleerde selproliferasie
nie. Wortmannin het geen effek gehad op insulien- of20% FKS-gestimuleerde DNA sintese nie,
maar het wel die aktivering van PB-kinase se metaboliese teiken, AktJPKB geinhibeer. Insulien
seintransduksie aktiveer dus ERK deur beide MEK en PB-kinase, maar het geen effek op
proliferasie gehad nie. FKS-gestimuleerde ERK aktivering en proliferasie was totaal afhanlik
van MEK-ERK aktivering. Insulien-geaktiveerde DNA sintese in die mens MG-63 osteoblaste was gedeeltelik afhanklik
van beide MEK en PB-kinase. Alhoewel IPA ook PB-kinase kon aktiveer, was dit totaal
afhanklik van MEK vir DNA sintese. Dit dui aan dat daar 'n PB-kinase stroom-op van 'n
dominante MEK-ERK seintransduksie pad voorkom. Die verskille wat ons dus waargeneem het
in insulien mitogeniese seintransduksie tussen muis en mens, kan aandui dat insuliengestimuleerde
seintranduksie paaie kan verskil van spesie tot spesie. Dit is bevestig met die
muisselle wat onafhanklik is en mens selle wat afhanklik is van MEK aktivering vir insuliengeaktiveerde
DNA sintese.
Kroniese, langtermyn steroied behandeling kan beenverlies veroorsaak en die effek van
glukokortikoide (GK) op die insulien mitogeniese pad in osteoblaste is dus ook ondersoek.
Natrium-ortovanadaat, 'n proteien tirosien fosfatase (PIP) inhibitor het GK-verlaagde
proliferasie in repons tot beide IPA- en FKS behandeling herstel in MBA-lSA en MG-63
preosteoblaste. PIPs soos SHP-l en PIP-l B funksioneer deur gefosforileerde kinases te
defosforileer en dus te inaktiveer. Beide SHP-l and PIP-lB kon assosieer met kinases in die
mitogeniese insulien seintransduksie pad van vinnig groeiende MBA-IS A preosteoblaste in
10% FKS. Verder het SHP-I ook geko-immunopresipiteer met aktiewe, tirosien-gefosforileerde
ERK, wat aandui dat SHP-I met ERK assosieer om dit te defosforileer en inaktiveer. Die MEKERK
of PB-kinase paaie is nie belangrik vir insulien-geaktiveerde seintransduksie in muis
osteoblaste nie. Dit is dus onwaarskynlik dat die PIPs 'n rol sal speel in die negatiewe
regulering van hierdie seintransduksie paaie. Die ontdekking dat vanadaat nie glukokortikoiedverlaagde
insulien-geaktiveerde DNA sintese kan herstel nie, toon dat vanadaat-sensitiewe PIPs
nie 'n rol speel in insulien-geaktiveerde proliferasie in muisselle nie. Hierdie bevinding het
verder bevestig dat 'n nuwe insulien mitogeniese pad in die MBA-ISA, maar nie die MG-63
selle moontlik bestaan.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52646 |
| Date | 03 1900 |
| Creators | Langeveldt, Carmen Ronel |
| Contributors | Hulley, P. A., Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 88 p. : ill. |
| Rights | Stellenbosch University |
Page generated in 0.0029 seconds