Selective inhibition of rat mesangial cell proliferation by a synthetic peptide derived from the sequence of the C2 region of PKCβ

Abstract

RACK (receptor for activated C-kinase) is a protein that binds and translocates protein kinase C (PKC) to the appropriate cellular organelles. The binding of RACK has been mapped to C2 region of PKC. A number of peptides from the C2 region of PKCβ have been shown to inhibit the translocation and activation of PKCβ. This investigation was undertaken to study the role of PKCβ in rat mesangial cell proliferation mediated by a number of mitogens. Exposure of rat mesangial cells to thrombin, endothelin, epidermal growth factor, and phorbol 12,13-dibutyrate resulted in increased [³H]thymidine incorporation. Pretreatment of mesangial cells with Ro 32–0432 (selective PKC inhibitor) inhibited the proliferation mediated by all the above mitogens, suggesting that these mitogens mediated proliferation through PKC. Experiments were performed to further evaluate the involvement of PKCβ in this process by using the peptide derived from the C-2 region of PKCβ as a tool. The data suggest that although the peptide (P) alone had no effect on basal- or mitogen-mediated proliferation, the peptide in the presence of a carrier peptide (PC) inhibited proliferation mediated by endothelin. In the same experiment, proliferation mediated by epidermal growth factor, thrombin and phorbol dibutyrate was unaffected, suggesting that in rat mesangial cells, endothelin mediated proliferation through the activation of PKCβ.

Introduction

Protein kinase C (PKC), originally discovered as Ca²⁺- and phospholipid-dependent enzyme [6], is a family of at least 11 isozymes that plays a key role in the regulation of a number of cellular functions by phosphorylating appropriate cellular proteins. The PKC isozymes are divided into three groups based on the activator requirement. They are: 1) conventional or cPKC (α, β1, β2, and γ) that require Ca²⁺, negatively charged phospholipids, and diacylglycerol or phorbol ester for maximum activation; 2) novel or nPKC (δ, ϵ, σ, η, and μ) that require negatively charged phospholipids and diacylglycerol or phorbol ester, but not Ca²⁺, for activation; and 3) atypical or aPKCs (λ and ζ) that do not require Ca²⁺ or diacylglycerol but require negatively charged phospholipids for activation [5], [7]. All PKC isozymes contain unique stretches of sequences called variable or V regions (V1-V5) interspersed with sequences that are common to all isozymes and are called C regions (C1-C4). The differences between the three groups of isozymes are found in the common regions within the regulatory domains. Whereas the conventional or cPKC isozymes contain C1 and C2 regions at their regulatory domain, nPKC contain only C1 region, suggesting that C2 domain confers calcium sensitivity to the enzyme [2], [5], [7], [13], [17], [18].

Activation of PKC has been shown to involve translocation of the enzyme to the appropriate cell organelle. Elegant studies conducted by Daria Mochly Rosen and colleagues have identified proteins that specifically bind PKC in the presence of its activators, and translocate the enzyme to the cell particulate fraction [10], [11], [12], [15]. These proteins are called receptors for activated C kinases (RACKs), and cloning of RACK1 has been reported by Ron et al. [14].

The RACK binding site has been localized at the C2 region of PKC [15]. Three peptides of 8–13 amino acids long, synthesized based on the C2 region of PKCβ, inhibited the binding of RACK1 to C2 containing PKC as well as the translocation and function of PKCβ in vivo [15]. In the same experiments, a number of control peptides failed to inhibit the binding of RACK1 to PKC or the function of PKC [15].

Mesangial cell proliferation is a key feature in glomerulonephritis, and studies have indicated that PKCβ2 is at least partially responsible for this growth [3], [4], [16]. Rat renal mesangial cells have been shown to proliferate in response to a number of growth factors as well as ligands that act through 7 transmembrane G protein-coupled receptors. In the present investigation, we used a C2 region-derived peptide of PKCβ to study the involvement of this enzyme in mesangial cell proliferation. Because peptides cannot cross the cell membrane to get to the inside of the cells, we took the approach of delivering this peptide by using a carrier peptide that is the hydrophobic region of the signal peptide and is shown to interact with lipid bilayers [19]. By using this procedure, Lin et al. [9] have shown the inhibition of the nuclear translocation of NFkB by a synthetic peptide containing a cell membrane permeable motif. This technique eliminates the need for permeabilization of cells to deliver peptides to the inside of the cell. The data presented here clearly demonstrate that the peptide derived from the C2 region of PKCβ was effective only when it was delivered along with the carrier peptide. In addition, it inhibited specifically endothelin (ET)-mediated proliferation without affecting thrombin, epidermal growth factor (EGF), or phorbol 12,13-dibutyrate (PDBu)-mediated proliferation. Under the same conditions, the peptide without carrier peptide did not have any effect.