Pharmacological inhibition of PKC and buffering of intracellular
calcium affects the hematopoiesis-supportive ability of TGF-primed
BMSCs.
TGFβ1 is known to activate eNOS via activation of PKC [22]. An
increase intracellular calcium [Ca2+]i is also
known to activate eNOS [23]. In my earlier study, I have shown that
treating BMSCs with TGFβ1 boosts their hematopoiesis-supportive ability
[18]. To determine whether a pharmacological inhibition of these two
effectors could interfere with the action of TGFβ1on the BMSCs, I
treated BMSCs with a cell-permeant octapeptide PKC inhibitor (Bachem,
10µg/ml) [24]and a cell-permeant chelator of
[Ca2+]i BAPTA-AM (Molecular Probes, 10µg/ml) 1 hr.
before applying TGFβ1 (Sigma, 10ng/ml; BMSC*TGF) to them. The plates
were incubated overnight at 37°C/5%CO2. After gently
washing the treated BMSCs with plain medium, 1 X 106human BM-derived MNCs were interacted with them for 1 hr., and then the
nonadherent cells were gently washed. The cells closely interacting with
the stromal layer were subjected to CFU assay as described [18].
This protocol has been used in all subsequent CFU-related experiments.
I found that the MNCs interacting with BMSC*TGF pretreated with the PKC
inhibitor or BAPTA-AM formed a significantly lower number of colonies
than those interacting with BMSC*TGF (Fig. 1a, 1b; 2ndand 3rd bars). As seen in my earlier study [18],
the MNCs interacting with BMSC*TGF formed significantly more colonies
than those interacting with control BMSCs (Fig. 1a,1b;
1st and 2nd bars).
Next, I also examined the effect of such inhibition on the types of CFU
formed. A differential count of colonies belonging to BFU-E, GM, and
GEMM revealed that the effect was across all colonies formed (Fig. 1c).
There was no bias towards any particular type of colony.
I further examined the effect of these BMSCs on CD34+HSCs isolated from the BM MNCs. As described earlier [18], I
co-cultured human BM-derived CD34+ cells with these
variously treated BMSCs, and after 48 hrs., immuno-stained them with an
antibody to CD34. As seen in Fig. 1d, the pre-treatment of BMSC*TGF with
the PKC inhibitor and BAPTA-AM completely inhibited the expansion of
CD34 HSCs. As expected, CD34+ cells co-cultured with
BMSC*TGF proliferated luxuriously compared to those co-cultured with
control BMSCs [18].
These data show that the proliferation of CD34+ HSCs
in response to the interaction with TGF-primed BMSCs critically involves
activated PKC and increased intracellular calcium levels.