T-kininogen can either induce or inhibit proliferation in Balb/c 3T3 fibroblasts, depending on the route of administration M. Aravena a , C. Pe ´rez a , V. Pe ´rez a , C. Acun ˜a-Castillo a , C. Go ´mez a , E. Leiva-Salcedo a , S. Nishimura a , V. Sabaj a , R. Walter b , F. Sierra b, * a Programa de Biologı ´a Celular y Molecular, Facultad de Medicina, Instituto de Ciencias Biome ´dicas, Universidad de Chile, Independencia 1027, Santiago, Chile b Lankenau Institute for Medical Research, 100 Lancaster Avenue, Wynnewood, PA 19096, USA Received 27 July 2004; received in revised form 27 August 2004; accepted 14 September 2004 Available online 13 October 2004 Abstract T-kininogen (T-KG) is a precursor of T-kinin, the most abundant kinin in rat serum, and also acts as a strong and specific cysteine proteinase inhibitor. Its expression is strongly induced during aging in rats, and expression of T-KG in Balb/c 3T3 fibroblasts results in inhibition of cell proliferation. However, T-KG is a serum protein produced primarily in the liver, and thus, most cells are only exposed to the protein from the outside. To test the effect of T-KG on fibroblasts exposed to exogenous T-KG, we purified the protein from the serum of K- kininogen-deficient Katholiek rats. In contrast to the results obtained by transfection, exposure of Balb/c 3T3 fibroblasts to exogenously added T-KG leads to a dose-dependent increase in [ 3 H]-thymidine incorporation. This response does not require kinin receptors, but it is clearly mediated by activation of the ERK pathway. As a control, we repeated the transfection experiments, using a different promoter. The results are consistent with our published data showing that, under these circumstances, T-KG inhibits cell proliferation. We conclude that T-KG exerts opposite effects on fibroblast proliferation, depending exclusively on the way that it is administered to the cells (transfection versus exogenous addition). # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Aging; Kininogen; Fibroblasts; Proliferation; Signal transduction 1. Introduction T-kininogen (T-KG) is a multifunctional protein char- acterized primarily as a precursor of the vasoactive peptide T-kinin and as a potent physiological inhibitor of cysteine proteinases (Anderson and Heath, 1985). Hepatic expression of the T-KG gene increases dramatically towards the end of lifespan in rats of different strains and of both sexes (Sierra et al., 1989; Walter et al., 1998). This in turn leads to an increase in serum levels of the protein (Sierra et al., 1992), and we have recently shown that serum kinin levels are also increased in parallel (V. Pe ´rez et al., in preparation). Through differential splicing, the other major kininogen in the rat, K-kininogen, gives rise to both HMW- and LMW- KG (Kakizuka et al., 1988), and we have also previously shown an age-related increase in serum levels of HMW-KG in rats (Sierra, 1995). Similar observations have been made for the human ortholog, HMW-KG (Kleniewski and Czokalo, 1991). In order to examine the physiological role of the increase in T-KG during aging, we have previously expressed the protein in several fibroblast cell lines. When using the strong constitutive CMV promoter, T-KG expression was incompatible with cell growth (unpublished data), while more modest expression under control of the metallothionein-1 promoter resulted in a strong inhibition of proliferation in both Balb/c 3T3 and L TK fibroblasts (Torres et al., 2001). This inhibition was accompanied by a decrease in basal ERK activity, apparently as a result of T-KG- www.elsevier.com/locate/mechagedev Mechanisms of Ageing and Development 126 (2005) 399–406 Abbreviations: ERK, extracellular signal-regulated kinase; [ 3 H]-TdR, [3H]-thymidine; HMW-KG, high-molecular-weight kininogen; LMW-KG, low-molecular-weight kininogen; T-KG, T-kininogen * Corresponding author. Tel.: +1 301 496 6402; fax: +1 301 402 0010. E-mail address: [email protected] (F. Sierra). 0047-6374/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2004.09.006
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T-kininogen can either induce or inhibit proliferation in Balb/c
3T3 fibroblasts, depending on the route of administration
M. Aravenaa, C. Pereza, V. Pereza, C. Acuna-Castilloa, C. Gomeza,E. Leiva-Salcedoa, S. Nishimuraa, V. Sabaja, R. Walterb, F. Sierrab,*
aPrograma de Biologıa Celular y Molecular, Facultad de Medicina, Instituto de Ciencias Biomedicas, Universidad de Chile,
Independencia 1027, Santiago, ChilebLankenau Institute for Medical Research, 100 Lancaster Avenue, Wynnewood, PA 19096, USA
Received 27 July 2004; received in revised form 27 August 2004; accepted 14 September 2004
Available online 13 October 2004
www.elsevier.com/locate/mechagedev
Mechanisms of Ageing and Development 126 (2005) 399–406
Abstract
T-kininogen (T-KG) is a precursor of T-kinin, the most abundant kinin in rat serum, and also acts as a strong and specific cysteine
proteinase inhibitor. Its expression is strongly induced during aging in rats, and expression of T-KG in Balb/c 3T3 fibroblasts results in
inhibition of cell proliferation. However, T-KG is a serum protein produced primarily in the liver, and thus, most cells are only exposed to the
protein from the outside. To test the effect of T-KG on fibroblasts exposed to exogenous T-KG, we purified the protein from the serum of K-
kininogen-deficient Katholiek rats. In contrast to the results obtained by transfection, exposure of Balb/c 3T3 fibroblasts to exogenously added
T-KG leads to a dose-dependent increase in [3H]-thymidine incorporation. This response does not require kinin receptors, but it is clearly
mediated by activation of the ERK pathway. As a control, we repeated the transfection experiments, using a different promoter. The results are
consistent with our published data showing that, under these circumstances, T-KG inhibits cell proliferation. We conclude that T-KG exerts
opposite effects on fibroblast proliferation, depending exclusively on the way that it is administered to the cells (transfection versus exogenous
addition).
# 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Aging; Kininogen; Fibroblasts; Proliferation; Signal transduction
1. Introduction
T-kininogen (T-KG) is a multifunctional protein char-
acterized primarily as a precursor of the vasoactive peptide
T-kinin and as a potent physiological inhibitor of cysteine
proteinases (Anderson and Heath, 1985). Hepatic expression
of the T-KG gene increases dramatically towards the end of
lifespan in rats of different strains and of both sexes (Sierra
et al., 1989; Walter et al., 1998). This in turn leads to an
increase in serum levels of the protein (Sierra et al., 1992),
and we have recently shown that serum kinin levels are also
with T-KG alone. HOE: HOE-140, DesArg: Des-Arg9-Leu8-BK, PD:
PD098056, Bis: bis-indoleylmaleimide.
On the other hand, Fig. 4B indicates that the effect of
2 mg/ml T-KG on [3H]-TdR incorporation is completely
abrogated in the presence of the MEK inhibitor PD098056,
and partially eliminated after inhibition of the PKC path-
way (by bis-indoleylmaleimide). In contrast, the stimula-
tory effect was not significantly affected by an inhibitor
of the PKA pathway, H89. These results indicate that
entry into S phase by the addition of exogenous T-KG
requires activation of the ERK pathway of signal trans-
duction, via a mechanism that does not involve the kinin
receptors.
3.5. Exogenous T-KG induces ERK activation and
synthesis of cyclin A
The involvement of ERK and entry of the cells into S
phase was further confirmed by directly measuring ERK
activation and cyclin A synthesis in response to T-KG. Fig.
5A shows that exogenous addition of T-KG results in ERK
phosphorylation, with both kinetics and strength comparable
to those observed after serum stimulation. Consistent with
this result, Balb/c 3T3 fibroblasts started synthesis of cyclin
A at approximately 16 h after treatment with 2 mg/ml T-KG
(Fig. 5B). Again, this result is both quantitatively and
kinetically comparable to serum stimulation by 10% FBS.
These observations further confirm the data from [3H]-TdR
incorporation experiments, which indicate that exogenous T-
KG can induce entry into S phase or DNA synthesis in
cultured fibroblasts.
4. Discussion
We have previously reported that expression of T-
kininogen is considerably increased in the liver of old rats
(Sierra et al., 1989), leading to increased serum levels (Sierra
et al., 1992). Our previous work has also shown that T-KG
can dramatically inhibit cell proliferation when expressed
endogenously in Balb/c 3T3 or LTK� fibroblasts (Torres et
al., 2001). This inhibition appears to be the result of
decreased ERK activity, as a consequence of stabilization of
the phosphatase MKP-1 (Torres et al., 2000, 2001). While
the protein has been detected in a variety of tissues (Chao et
al., 1988; Damas et al., 1992; Gao et al., 1992; Oza et al.,
1990), its mRNA has been observed primarily in hepato-
cytes. Thus, even though fibroblasts can express T-KG in
response to cAMP, prostaglandin E2 and other cytokines
(Takano et al., 1995), ectopic expression of the protein in
these cells is not physiologically relevant. Fibroblasts are not
normally exposed to serum proteins either, but this can
happen under pathological conditions where there is either
plasma transvasation or a rupture of the endothelial layer.
Thus, fibroblasts can be viewed as a valid model for
exogenous exposure of cells to circulating T-KG under
pathological conditions, and perhaps during aging. There-
fore, we decided to further test the possibility that T-KG
M. Aravena et al. / Mechanisms of Ageing and Development 126 (2005) 399–406404
Fig. 5. T-KG induces ERK activation and cyclin A synthesis in Balb/c 3T3 fibroblasts. Cells were seeded and serum-starved for 48 h as before, and then they
were stimulated either by 10% BFS or by 2 mg/ml T-KG. (A) ERK phosphorylation (P-ERK) was measured at short times after induction (up to 2 h). (B) Cyclin
A accumulation was measured at times up to 24 h. Western blot analysis was performed and representative blots are shown, probed for phosphorylated ERK (P-
ERK) and total ERK (ERK-1) (A), as well as cyclin A and b-actin (B).
might inhibit entry into DNA synthesis when added
exogenously to these cells.
Our preliminary experiments were surprising, as we
observed an induction of [3H]-TdR incorporation, exactly
the opposite of what we had expected. Nevertheless, the
effects were very reproducible, observed under both
quiescent (low serum) and logarithmic growth conditions,
and they were concentration-dependent within the sub-
physiological range of concentrations tested. Interestingly,
we had observed a similar result before, by using
conditioned medium from the transfected cells (FS,
unpublished data). Due to the untidy nature of these early
experiments, they were not pursued further at the time, and
instead, purified T-KG was used for all further experiments.
Thus, while it is still theoretically possible that an extremely
powerful minor contaminant in our preparation could be
responsible for the effects we have observed, these
experiments suggest that the same molecule can indeed
produce both a pro- and an anti-proliferative effect,
depending on which side of the plasma membrane it finds
itself. One possible explanation for the discrepancy with our
previous results is cell drifting. To test this possibility, we
repeated our previous transfection studies, using the same
batch of cells we were using for the exogenous application
experiments. Even though we used a different promoter,
these experiments resulted in confirmation of the previous