2.2 Methods:
2.2.8 Next generation sequencing
2.2.8.5 Sequencing
2.2.8.5.1 Chip preparation
Ion Proton chip adaptor was attached to the chip exit well. 200µL of
100% isopropanol was injected into the chip loading port; the expelled liquid
was removed from the exit well. The isopropanol was aspirated from the chip
for 5-10 secs to ensure that the chip is dry. 100µL of Ion PI chip preparation
solution was injected into the chip loading port; the expelled liquid was removed
from the exit well. The chip was place on a 50°C heat block for 2 mins. 200µL
of 100% isopropanol was injected into the chip loading port; the expelled liquid
was removed from the exit well. This was repeated a total of two times. 200µL
of nuclease free water was injected into the chip loading port; and the expelled
liquid removed. 200µL of 0.1M NaOH was then injected into the chip loading
port, and the expelled liquid was removed from the exit well. The chip was
incubated at room temperature for 1 min then 200µL of nuclease free water was
injected into the chip loading port. These steps were repeated a total of three
times. 200µL of 100% isopropanol was injected into the chip loading port; the
expelled liquid was removed from the exit well. 100µL of 100% isopropanol
was pipetted onto the chip loading well, the liquid was then removed from the
same well. The Ion Proton chip adapter was removed from the chip.
2.2.8.5.2 Loading of sample on the chip
centrifuged for 30 secs in the Ion Chip Minifuge followed by injection of 100µL
of foaming solution and then centrifuged again for 30 secs. The chip was
injected with 100µL of the flushing solution 2 times; the expelled solution was
discarded after each injection. 100µL of 50% annealing buffer was injected into
the chip loading port three times; the expelled liquid was removed between each
injection. 65µL of polymerase solution (6µL Ion PI Sequencing polymerase
with 60µL 50% annealing buffer) was injected; the expelled liquid was
removed. The chip was left to incubate for 5 mins then secured in the Ion Proton
sequencer.
2.2.9 Nucleofection using siRNA
CASMCs were seeded at 100,000 cells/well (5mM DMEM, 10% FBS,
no antibiotics) in a 24 well plate (Nunc). The cells were incubated at 37oC in 5%
CO2 until they reached 70-80% confluency then serum deprived (5mM DMEM,
0.1% FBS, no antibiotics) for 24 hours prior to Nucleofection. Using the Lonza
AD1 4D-Nucleofector Y kit, 1mL of the supplement was mixed with 4.5mL of
AD1 4D-Nucleofector solution. 105pmol of siRNA was made up with 350µL
Nucleofector solution. The media was carefully removed and 350µL of the
siRNA substrate solution was carefully transferred into each well. The 24 well
dripping electrode array was inserted into the 24 well plate. The Nucleofection
process began when the plate was placed in 4D-Nucleofecor core Y-unit using
ER137 pulse. The 24 well dipping electrode array was carefully removed from
24 well plate. The siRNA substrate solution was removed from each well and
immediately replaced with 1mL of pre-warmed medium (5mM DMEM,
stated otherwise) before they were treated with agonists to assess mRNA
expression.
2.2.10 Statistical Analysis
Data was normalised and is shown as the mean ± standard error of the mean of three independent experiments, unless stated otherwise. A one-way ANOVA was used to calculate statistical significance of normalised data as stated followed by least significant difference post-hoc analysis. Results were considered significant when the probability was less than 0.05 and 0.01.
Chapter-3. Proteinase activated
receptor-1 mediated dual kinase
receptor transactivation
stimulates the expression of
glycosaminoglycan synthesizing
3.1 Introduction
Seven transmembrane GPCRs are the largest group of cell surface
receptors in biology. GPCR signalling involves the traditional cascade which
will be referred to as transactivation-independent signalling (Kamato et al.,
2014b) or GPCR mediated transactivation of cell surface PTKR. Two decades
ago the initial finding of transactivation described GPCR activation of the
PTKR, EGFR, stimulating the immediate downstream products phospho-Erk1/2
(Daub et al., 1996). This discovery greatly expanded the known functions of
GPCRs.
The paradigm of GPCR mediated kinase receptor transactivation has
been expanded to include GPCR mediated activation of S/TKR, in particular the
TGFBR1 (Burch et al., 2010, Burch et al., 2013, Little et al., 2010, Ivey and
Little, 2008). PAR-1 transactivation of the TGFBR1 is studied by the
phosphorylation of the immediate downstream intermediate of TGFBR1, Smad2
in the carboxy terminal (phospho-Smad2C) (Burch et al., 2010, Little et al.,
2010). Thrombin stimulation of human VSMCs leads to the synthesis of the
ECM molecules proteoglycans. Recently our lab reported that thrombin
treatment of VSMCs leads to an increase in radio sulphate incorporation into
secreted proteoglycans a response which is approximately equally blocked by
antagonists of the EGFR (AG1478) and the TGFBR1 (SB431542). The two
blocked by the PAR-1 antagonist SCH79797 (Burch et al., 2010, Burch et al.,
2013).
Previous work has shed light on the specific enzymes which mediate the
synthesis of CS/DS GAG chains on proteoglycans (Sugahara and Kitagawa,
2000, Silbert and Sugumaran, 2002). The synthesis of CS/DS GAG chains on
proteoglycans involves the concerted action of a number of GAG synthesizing
genes (Mikami and Kitagawa, 2013). The growth factor and hormone signalling
pathways that control the expression of these enzymes are important because
GAG hyperelongation is a critical step in the early development of
atherosclerosis (Ivey et al., 2008, Getachew et al., 2010a, Ivey and Little, 2008).
During the progression of atherosclerosis, the elongation of CS chains in the
arterial wall involves at least four GAG synthesizing genes specifically
CHST11, CHSY1, CHPF, and chondroitin N-acetylgalactosaminoglycan-2
(Anggraeni et al., 2011) (Refer to Chapter 1 Section 1.3 for more details).
Increased LDL binding in the 8 weeks of the development of atherosclerosis
was associated with an elevated mRNA expression of the four genes. CHST11
and CHST3 are sulfotransferases whereas CHPF and CHSY1 are
glycosyltransferases which are directly involved in the synthesis of the GAG
chains thus the regulation of proteoglycan size (Izumikawa et al., 2011). As
such, the signalling pathways leading to the expression of these GAG
synthesizing enzymes represent potential therapeutic targets for the treatment of
atherosclerosis.
Proteoglycans secreted by VSMCs in vivo are present in early
atherosclerotic lesions (Nakashima et al., 2007). Thrombin treatment of VSMCs
leads to the hyperelongation of GAG chains of proteoglycans such as biglycan
described to be involved in this signalling cascade, however the downstream
gene targets presumably the specific GAG synthesizing enzymes that are
involved remain unknown. This chapter seeks explores the role of
transactivation dependent signalling pathways that mediated GAG gene
expression.