Despite our previous studies on signaling pathways that mediate hypoxia-induced proliferation of fibroblasts (Das et al., 2000
), it remains unclear how replication suppressors, which simultaneously coexist with proliferative stimulators, work collaboratively to attenuate normal proliferation of hypoxic fibroblasts. In the present study, we report for the first time that PKCζ is the master regulator of hypoxia-induced ERK1/2 dephosphorylation events through the regulation of MKP-1 expression and thereby limits proliferation of hypoxic fibroblasts (). PKCζ attenuation leads to striking up-regulation in proliferation as well as ERK1/2 phosphorylation in hypoxic fibroblasts. In contrast, PKCζ overexpression induces a significant down-regulation of replication in hypoxic cells. PKCζ regulates hypoxia-induced MKP-1 expression in fibroblasts. MKP-1 blockade mimics the results of PKCζ attenuation on hypoxia-stimulated ERK1/2 phosphorylation and proliferation. These results strongly support the idea that PKCζ acts as a replication repressor through its regulation of MKP-1 expression in hypoxic fibroblasts.
Schematic diagram: role of PKCζ in the termination of proliferation of hypoxic fibroblasts through the regulation of ERK1/2 dephosphorylation and MKP-1 expression.
Our data in vascular fibroblasts contrast with the majority of published reports where PKCζ is described as a proliferative mediator in a variety of cells (Hirai and Chida, 2003
). Recently, Braun and Mochly-Rosen (2003
) have demonstrated that PKCζ is required for TGFβ1-induced proliferation of neonatal primary cardiac fibroblasts. PKCζ attenuation also inhibits platelet-derived growth factor (PDGF)-induced proliferation of human airway smooth muscle cells (Carlin et al., 2000
). However, a recent report also demonstrates that PKCζ blockade does not inhibit rabbit vascular smooth muscle cell proliferation (Hussain et al., 2002
). In fact, PKCζ inhibition increases growth factor- and cytokine-induced proliferation, which supports the notion that PKCζ is an antiproliferative kinase under specific circumstances and is in agreement with our results describing the role of PKCζ as proliferative repressor of hypoxic fibroblasts. Therefore, the role of PKCζ in proliferation is not only cell type specific, but also species specific and hence, the function of this particular atypical isozyme in cellular proliferative responses requires more rigorous characterization.
PKCζ plays an important functional role in mitogenic signaling by initiating the activation of the downstream MAP kinases such as MEK and ERK family proteins (Berra et al., 1993
). Activation of the ERK cascade is known to be associated with cellular proliferation (Chang et al., 2003
). Previously, we have also reported that proliferation of hypoxic fibroblasts is regulated by hypoxia-induced ERK1/2 activation (Short et al., 2004
). However, in the present study, PKCζ attenuation results in persistent ERK1/2 phosphorylation, which contributes to the exuberant proliferation of hypoxic fibroblasts. Another important point is that PKCζ blockade induces accumulation of activated ERK1/2 in the nucleus, which is consistent with the fact that presence of activated ERK1/2 in the nucleus is necessary for the induction of cell proliferation (Pouyssegur et al., 2002
). PKCζ-induced termination of ERK phosphorylation observed in the present studies is in contrast to the previously published reports demonstrating that PKCζ is the upstream kinase of MEK and ERK in other cell types (Berra et al., 1993
). Also, in spite of the similar increases in proliferation of our cells and rabbit vascular smooth muscle cells (Hussain et al., 2002
) upon PKCζ inhibition, the role of PKCζ in ERK1/2 activation is different between the two cell types. In the vascular fibroblasts, PKCζ is the master regulatory switch of ERK1/2 dephosphorylation events, whereas in rabbit smooth muscle cells ERK1/2 activation is independent of PKCζ activation (Hussain et al., 2002
). Therefore, the functional role of PKCζ in the regulation of ERK1/2 activation upon a stimulus is a highly cell- and condition-specific event.
Because of the critical importance of ERK1/2 in cellular signaling, the activities of ERKs must be tightly regulated and this can be achieved by ERK-specific phosphatases, e.g., MKP family members. There are at least 11 MKPs in mammals, which imply the existence of a complex regulatory network for MAP kinase signaling. MKPs differ by properties such as tissue-specific expression, differential regulation in response to various stimuli, distinct subcellular localization and substrate specificity. MKP-1 has also been identified as a hypoxia-responsive gene in a variety of cell types (Keyse and Emslie, 1992
; Noguchi et al., 1993
; Laderoute et al., 1999
) and is an immediate-early gene product, characterized by rapid transcriptional induction after MAP kinase family activation, short half-life, and rapid destruction by the 26S proteasome (Keyse and Emslie, 1992
; Keyse, 1995
). MKP-1 participates in the determination of the kinetics and thus the cellular outcome of MAP kinase signaling (e.g., proliferation vs. differentiation; Charles et al., 1993
; Nishida and Gotoh, 1993
; and survival vs. apoptosis; Xaus et al., 2001
). The interaction between MKP-1/MAP kinase signaling also exhibits cell type specificity (Zhang et al., 2003
In the present study, hypoxia-induced up-regulation of MKP-1 provides a novel mechanism, to account for the inhibitory effects of PKCζ on ERK1/2 phosphorylation and proliferation in hypoxic fibroblasts. Marked prolongation of ERK1/2 phosphorylation in cells treated with either PKCζ antisense oligonucleotides or myristoylated PKCζ peptide inhibitor correlates with the inhibition of MKP-1 expression upon PKCζ attenuation. Consequently, our results allow us to conclude that PKCζ is the suppressor of ERK1/2 phosphorylation by regulating MKP-1 expression. Prolonged activation of PKCζ in hypoxic fibroblasts () might be required by the system to maintain the level of MKP-1 upon hypoxic exposure. MKP-1 overexpression inhibits ERK-regulated reporter gene expression, Ras-induced DNA synthesis, and growth-factor-stimulated entry into the S phase in fibroblasts (Brunet et al., 1995
). As a result, MKP-1 expression constitutes a control mechanism for attenuation of mitogenic signaling pathways. Our data further suggest that PKCζ-mediated MKP-1 expression plays a crucial role in the proper termination of ERK1/2 activation during the hypoxic exposure and contributes to “switching off” the signals directing the proliferation of hypoxic fibroblasts.
Evidence is accumulating to indicate that PKC is associated with nuclear events both in resting cells as well as in actively dividing cells (Capitani et al., 1987
; Chiarugi et al., 1990
; Buchner et al., 1992
; Neri et al., 1994
). In the present study, PKCζ localization in the nuclear compartment is consistent with the report demonstrating its expression in the nuclei of unstimulated adipocytes (Lacasa et al., 1995
). Stimulation of the adipocytes with insulin or serum caused a rapid increase in nuclear PKCζ activity, suggesting that PKCζ could directly phosphorylate structural and/or regulatory nuclear proteins. Nucleolin and heterogeneous nuclear ribonucleoprotein are the substrates of PKCζ, suggesting that PKCζ may play an important role in nuclear signal transduction (Tuteja and Tuteja, 1998
). Nuclear PKCζ might be the critical mediator of the hypoxic responses in fibroblasts because PKCζ transactivates HIF-1α by blocking the expression of a factor inhibiting HIF-1 in renal cancer cells (Datta et al., 2004
). Interestingly, MKP-1 colocalizes with PKCζ in the nuclear compartment of fibroblasts. PKCζ association with MKP-1 in the nucleus may constitute the critical replication repressor system in fibroblasts.
Manipulation of PKCζ levels, either by inhibition or overexpression, leads to subsequent alteration in MKP-1 levels, suggesting that PKCζ tightly regulates MKP-1 expression in fibroblasts. MKP-1 expression is also regulated by PKC, albeit by a different isozyme, PKCε, in bone marrow macrophages (Valledor et al., 2000
). In H41E rat hepatoma cells, insulin-induced MKP-1 expression is blocked by the myristoylated PKCζ pseudosubstrate peptide inhibitor (Lornejad-Schafer et al., 2003
). However, in that report PKCζ-mediated MKP-1 expression is not correlated with any cellular response. We believe that our data are the first demonstration of nuclear colocalization of MKP-1 and PKCζ, which together function as terminators of proliferative signals in hypoxic fibroblasts. Our future studies will focus on evaluating the mechanism of PKCζ-mediated regulation of MKP-1 expression in vascular fibroblasts.
Excessive proliferation of fibroblasts is associated with a number of vascular diseases (Stenmark et al., 1987
; Stenmark and Mecham, 1997
; Stenmark et al., 2000
; Rey and Pagano, 2002
), asthma, chronic obstructive pulmonary diseases, pulmonary fibrosis (Zhong et al., 2005
), and also cancer (Bhowmick et al., 2004
). Multiple factors including growth factors, cytokines, mechanical stress, hypoxia, neurotransmitters, and hormones are believed to contribute to the processes leading to fibroblast proliferation (Sartore et al., 2001
), wherein PKCζ might serve as a common second messenger mediating the termination signals for proliferative responses. One of the primary steps in the orchestrated “emergency stop” cascade may be the up-regulation of MKP-1 expression that dephosphorylates ERK1/2 and consequently attenuates proliferation. PKCζ exerts its suppressing effect on ERK activation and proliferation in vascular fibroblasts primarily through interactions that involve MKP-1. Thus, we postulate that the balance between PKCζ-mediated MKP-1 expression and ERK1/2 activities stimulated by hypoxia is critical for maintaining cellular homeostasis. Further understanding of the mechanisms by which hypoxia stimulates the PKCζ-mediated induction of MAP kinase phosphatase might lead to strategies for the prevention and treatment of fibroproliferative diseases resulting from chronic hypoxic exposure.