G. Duke Virca

The Soluble Form of IL-1 Receptor Accessory Protein Enhances the Ability of Soluble Type II IL-1 Receptor to Inhibit IL-1 Action

Regulation of the activity of the proinflammatory cytokine IL-1 is complex, involving transcriptional and translational control, precursor processing, a receptor antagonist (IL-1ra), and a decoy receptor. Here we report that the soluble form of the IL-1 receptor accessory protein (AcP) increases the affinity of binding of human IL-1alpha and IL-1beta to the soluble human type II IL-1 receptor by approximately 100-fold, while leaving unaltered the low binding affinity of IL-1ra. Soluble AcP is present in normal human serum at an average concentration greater than 300 ng/ml. These findings suggest that the soluble form of IL-1R AcP contributes to the antagonism of IL-1 action by the type II decoy receptor, adding another layer of complexity to the regulation of IL-1 action.

RIP4 Is an Ankyrin Repeat-Containing Kinase Essential for Keratinocyte Differentiation

The epidermis is a stratified, continually renewing epithelium dependent on a balance among cell proliferation, differentiation, and death for homeostasis. In normal epidermis, a mitotically active basal layer gives rise to terminally differentiating keratinocytes that migrate outward and are ultimately sloughed from the skin surface as enucleated squames. Although many proteins are known to function in maintaining epidermal homeostasis, the molecular coordination of these events is poorly understood. RIP4 is a novel RIP (receptor-interacting protein) family kinase with ankyrin repeats cloned from a keratinocyte cDNA library. RIP4 deficiency in mice results in perinatal lethality associated with abnormal epidermal differentiation. The phenotype of RIP4(-/-) mice in part resembles that of mice lacking IKKalpha, a component of a complex that regulates NF-kappaB. Despite the similar keratinocyte defects in RIP4- and IKKalpha-deficient mice, these kinases function in distinct pathways. RIP4 functions cell autonomously within the keratinocyte lineage. Unlike IKKalpha, RIP4-deficient skin fails to fully differentiate when grafted onto a normal host. Instead, abnormal hair follicle development and epidermal dysplasia, indicative of progression into a more pathologic state, are observed. Thus, RIP4 is a critical component of a novel pathway that controls keratinocyte differentiation.

Kallikrein-Related Peptidase 4: A New Activator of the Aberrantly Expressed Protease-Activated Receptor 1 in Colon Cancer Cells

Certain serine proteases are considered to be signaling molecules that act through protease-activated receptors (PARs). Our recent studies have implicated PAR1 and PAR4 (thrombin receptors) and PAR2 (trypsin receptor) in human colon cancer growth. Here we analyzed the expression of KLK4, a member of the kallikrein-related peptidase (KLK) family of serine proteases and explored whether this member can activate PAR1 and PAR2 in human colon cancer cells. Immunohistochemistry showed KLK4 expression in human colon adenocarcinomas and its absence in normal epithelia. KLK4 (1 micromol/L) initiated loss of PAR1 and PAR2 from the HT29 cell surface as well as increased intracellular calcium transients in HT29 cells. This KLK4-induced Ca2+ flux was abrogated after an initial challenge of the cells with TRAP (SFLLR-NH2; 100 micromol/L), which is known to desensitize PAR1 and PAR2. Interestingly, PAR1 blocking antibody, which inhibits cleavage and activation by thrombin, dramatically reduced KLK4-induced Ca2+ influx, but blocking cleavage of PAR2 failed to attenuate the KLK4-induced Ca2+ flux. Consistently, desensitization with AP1 (TFFLR-NH2), targeting PAR1, attenuated most of the Ca2+ flux induced by KLK4. KLK4 also induced a rapid and significant ERK1/2 phosphorylation in HT29 cells. Our results demonstrate, for the first time, that KLK4 is aberrantly expressed in colon cancer and capable of inducing PAR1 signaling in cancer cells. These data suggest that KLK4 signaling via PAR1 may represent a novel pathway in colon tumorigenesis.

Kallikrein-Related Peptidase 14 Acts on Proteinase-Activated Receptor 2 to Induce Signaling Pathway in Colon Cancer Cells

Serine proteinases participate in tumor growth and invasion by cleaving and activating proteinase-activated receptors (PARs). Recent studies have implicated PAR-1 and PAR-4 (activated by thrombin) and PAR-2 (activated by trypsin but not by thrombin) in human colon cancer growth. The endogenous activators of PARs in colon tumors, however, are still unknown. We hypothesize that the kallikrein-related peptidase (KLK) family member KLK14, a known tumor biomarker, is produced by colonic tumors and signals to human colon cancer cells by activating PARs. We found that i) KLK14 mRNA was present in 16 human colon cancer cell lines, ii) KLK14 protein was expressed and secreted in colon cancer cell lines, and iii) KLK14 (0.1 μmol/L) induced increases in intracellular calcium in HT29, a human colon cancer-derived cell line. KLK14-induced calcium flux was associated with internalization of KLK14-mediated activation of PAR-2. Furthermore, KLK14 induced significant extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation and HT29 cell proliferation, presumably by activating PAR-2. A PAR-2 cleavage and activation-blocking antibody dramatically reduced KLK14-induced ERK1/2 signaling. Finally, ectopic expression of KLK14 in human colon adenocarcinomas and its absence in normal epithelia was demonstrated by IHC analysis. These results demonstrate, for the first time, the aberrant expression of KLK14 in colon cancer and its involvement in PAR-2 receptor signaling. Thus, KLK14 and its receptor, PAR-2, may represent therapeutic targets for colon tumorigenesis.

Kallikrein-related peptidase signaling in colon carcinoma cells: targeting proteinase-activated receptors.

Abstract We hypothesized that kallikrein-related peptidase 14 (KLK14) is produced by colonic tumors and can promote tumorigenesis by activating proteinase-activated receptors (PARs). We found that KLK14 is expressed in human colon adenocarcinoma cells but not in adjacent cancer-free tissue; KLK14 mRNA, present in colon cancer, leads to KLK14 protein expression and secretion; and KLK14 signals viaPAR-2 in HT-29 cells to cause (1) receptor activation/internalization, (2) increases in intracellular calcium, (3) stimulation of ERK1/2/MAP kinase phosphorylation, and (4) cell proliferation. We suggest that KLK14, acting via PAR-2, represents an autocrine/paracrine regulator of colon tumorigenesis.