Quantitative RT-PCR analysis and immunohistochemical localization of the kallikrein-related peptidases 13 and 14 in lung

Expression of the KLK13 and KLK14 genes was examined at the mRNA and protein levels in a cohort of 57 patients with non-small-cell lung cancer (NSCLC). The mRNA levels, assessed by real-time RT-PCR, were significantly different in malignant tissues compared to adjacent non-malignant tissues (KLK13, p=0.006; KLK14, p=0.022). KLK13 and KLK14 mRNA overexpression in tumors (1/3 of the patients) was associated with a positive nodal status in multivariate analysis (p=0.018 and p=0.069, respectively). KLK13 and KLK14 were localized in the cytoplasm of epithelial cells of normal bronchus and NSCLC, as determined by immunohistochemistry. Moreover, positive staining was significantly associated with adenocarcinoma histotype (KLK13, p=0.014) and tumor size (KLK14, p=0.048). Although the results are marginally significant, patients with high KLK13 expression at the mRNA or protein level had lower overall survival.     

Mapping of the catalytic groove preferences of factor Xa reveals an inadequate selectivity for its macromolecule substrates

Factor Xa (FXa) hydrolyzes two peptide bonds in prothrombin having (Glu/Asp)-Gly-Arg-(Thr/Ile) for P(3)-P(2)-P(1)-P(1)' residues, but the exact preferences of its catalytic groove remain largely unknown. To investigate the specificity of FXa, we synthesized full sets of fluorescence-quenched substrates carrying all natural amino acids (except Cys) in P(3), P(2), P(1)', P(2)', and P(3)' and determined the k(cat)/K(m) values of cleavage. Contrary to expectation, glycine was not the "best" P(2) residue; peptide with phenylalanine was cleaved slightly faster. In fact, FXa had surprisingly limited preferences, barely more pronounced than trypsin; in P(2), the ratio of the k(cat)/K(m) values for the most favorable side chain over the least was 289 (12 with trypsin), but in P(1)', this ratio was only 30 (versus 80 with trypsin). This unexpected selectivity undoubtedly distinguished FXa from thrombin, which exhibited ratios higher than 19,000 in P(2) and P(1)'. Thus, with respect to the catalytic groove, FXa resembles a low efficiency trypsin rather than the highly selective thrombin. The rates of cleavage of the peptidyl substrates were virtually identical whether or not FXa was in complex with factor Va, suggesting that the cofactor did not exert a direct allosteric control on the catalytic groove. We conclude that the remarkable efficacy of FXa within prothrombinase originates from exosite interaction(s) with factor Va and/or prothrombin rather than from the selectivity of its catalytic groove.     

Dominant negative effectors of heparin affin regulatory peptide (HARP) angiogenic and transforming activities

Heparin affin regulatory peptide (HARP) is an heparin-binding growth factor, highly expressed in several primary human tumors and considered as a rate-limiting angiogenic factor in tumor growth, invasion, and metastasis. Implication of this protein in carcinogenesis is linked to its mitogenic, angiogenic, and transforming activities. Recently, we have demonstrated that the C-terminal residues 111-136 of HARP are required for its mitogenic and transforming activities (Bernard-Pierrot, I., Delbe, J., Caruelle, D., Barritault, D., Courty, J., and Milhiet, P. E. (2001) J. Biol. Chem. 276, 12228-12234). In this paper, HARP deleted of its last 26 amino acids was shown to act as a dominant negative effector for its mitogenic, angiogenic, transforming, and tumor-formation activities by heterodimerizing with the wild type protein. Similarly, the synthetic corresponding peptide P111-136 displayed in vitro inhibition of wild type HARP activities, but in this case, the inhibition was mainly explained by the competition of the peptide with HARP for the binding to the extracellular domain of the high affinity ALK receptor.     

Endothelial cell proliferation induced by HARP: implication of N or C terminal peptides

HARP (Heparin Affin Regulatory Peptide) is a 18-kDa secreted protein displaying high affinity for heparin. It has neurite outgrowth-promoting activity, while there are conflicting results regarding its mitogenic activity. In the present work, we studied the effect of human recombinant HARP expressed in bacterial cells as well as two peptides (HARP residues 1-21 and residues 121-139) on the proliferation of three endothelial cell types derived from human umbilical vein (HUVEC), rat adrenal medulla (RAME), and bovine brain capillaries (BBC) either added as a soluble form in the cell culture medium or coated onto the culture plate. HARP added in a soluble form in the culture medium had no effect on the proliferation of BBC, HUVEC, and RAME cells. However, when immobilized onto the cell culture plate, HARP had a concentration-dependent mitogenic effect on both BBC cells and HUVEC. The peptides presented as soluble factor induced a significant concentration-dependent mitogenic effect on BBC cells but only a small effect on HUVEC and RAME cells. When they were immobilized onto the cell culture plate, the mitogenic effect was much greater. The most responsive cells were BBC that expressed and secreted in the culture medium the higher amounts of HARP.     

Cell surface expression of melanocortin-1 receptor on HaCaT keratinocytes and alpha-melanocortin stimulation do not affect the formation and repair of UVB-induced DNA photoproducts.

Ultraviolet (UV) exposure induces an up-regulation of melanocortin-1 receptor (MC1R) expression in human skin and the alpha-melanocyte-stimulating hormone (alpha-MSH) may reduce UVB-induced DNA damage in normal human melanocytes. Using high-performance liquid chromatography coupled to tandem mass spectrometry, we investigated the formation and repair of DNA lesions in UVB-irradiated HaCaT cells stably transfected with the wild type MC1R gene (HaCaT-MC1R). Similar levels of 8 bipyrimidine photoproducts including cyclobutane pyrimidine dimers (CPDs) (T<>T, T<>C, C<>T), (6-4) photoproducts ((6-4)PPs) (TT-(6-4)PPs, TC-(6-4)PPs) and their Dewar valence isomers together with 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) were found to be generated in both non-transfected and HaCaT-MC1R cells after UVB exposure. Time-course studies of DNA photoproduct yields indicated that the DNA repair ability depended upon radiation doses. It was shown that (6-4)PPs were removed from the DNA of UVB-irradiated cells much more efficiently than CPDs. The repair efficiency of 8-oxodGuo, CPDs and (6-4)PPs was relatively similar in both cell lines and was not modified by stimulation with alpha-MSH before UVB-exposure. In conclusion, cell surface-enforced expression of MC1Rs on HaCaT keratinocytes and alpha-MSH stimulation do not affect the formation of UVB-induced DNA photoproducts and their subsequent repair.     

Biochemical comparative studies between eye- and brain-derived growth factors

Two bovine brain-derived growth factors, BDGF I and BDGF II, were isolated using the same extraction procedure as previously described for eye-derived growth factors (EDGF). The hypothesis that these growth factors were identical to EDGF I and EDGF II, respectively, was supported by their similar molecular weights (16,000 and 15,000, respectively) and isoelectric points (9.0 and 5.0, respectively), their identical retention behavior on reverse-phase chromatography and their similar amino acid compositions. From studies on their binding properties to cell surfaces, competition between EDGF I and BDGF I as well as competition between EDGF II and BDGF II to the same receptor was observed. The amino terminal sequence of EDGF II (1-16) was shown to be identical to the amino acid residues (7-22) of the acidic FGF, strongly confirming our observations on the identity of the factors isolated from bovine brain and retina.