Human DESC1 serine protease confers tumorigenic properties to MDCK cells and it is upregulated in tumours of different origin.

Proteolysis of the extracellular matrix components plays a crucial role in the regulation of the cellular and physiological processes, and different pathologies have been associated with the loss or gain of function of proteolytic enzymes. DESC1 (differentially expressed in squamous cell carcinoma gene 1), a member of the TTSP (type II transmembrane serine protease) family of serine proteases, is an epithelial-specific enzyme that has been found downregulated in squamous cell carcinoma of the head and neck region. We describe new properties of DESC1 suggesting that this protease may be involved in the progression of some type of tumours. Thus, this enzyme hydrolyses some extracellular matrix components, such as fibronectin, gelatin or fibrinogen. Moreover, Madin-Darby canine kidney (MDCK) cells expressing exogenous human DESC1 acquire properties associated with tumour growth such as enhanced motility and an increase of tubular forms in a 3D collagen lattice following HGF treatment. Finally, we generated polyclonal anti-DESC1 antibodies and immunohistochemical analysis in tissues different from head and neck region indicated that this protease was overexpressed in tumours of diverse origins. Taken together, our results suggest that DESC1 could be considered as a potential therapeutic target in some type of tumours.

Differential effects of amino-terminal distal and proximal domains in the regulation of human erg K(+) channel gating.

The participation of amino-terminal domains in human ether-a-go-go (eag)-related gene (HERG) K(+) channel gating was studied using deleted channel variants expressed in Xenopus oocytes. Selective deletion of the HERG-specific sequence (HERG Delta138-373) located between the conserved initial amino terminus (the eag or PAS domain) and the first transmembrane helix accelerates channel activation and shifts its voltage dependence to hyperpolarized values. However, deactivation time constants from fully activated states and channel inactivation remain almost unaltered after the deletion. The deletion effects are equally manifested in channel variants lacking inactivation. The characteristics of constructs lacking only about half of the HERG-specific domain (Delta223-373) or a short stretch of 19 residues (Delta355-373) suggest that the role of this domain is not related exclusively to its length, but also to the presence of specific sequences near the channel core. Deletion-induced effects are partially reversed by the additional elimination of the eag domain. Thus the particular combination of HERG-specific and eag domains determines two important HERG features: the slow activation essential for neuronal spike-frequency adaptation and maintenance of the cardiac action potential plateau, and the slow deactivation contributing to HERG inward rectification.

Modulation of human erg K+ channel gating by activation of a G protein-coupled receptor and protein kinase C.

1. Modulation of the human ether-à-go-go-related gene (HERG) K+ channel was studied in two-electrode voltage-clamped Xenopus oocytes co-expressing the channel protein and the thyrotropin-releasing hormone (TRH) receptor. 2. Addition of TRH caused clear modifications of HERG channel gating kinetics. These variations consisted of an acceleration of deactivation, as shown by a faster decay of hyperpolarization-induced tail currents, and a slower time course of activation, measured using an envelope of tails protocol. The voltage dependence for activation was also shifted by nearly 20 mV in the depolarizing direction. Neither the inactivation nor the inactivation recovery rates were altered by TRH. 3. The alterations in activation gating parameters induced by TRH were demonstrated in a direct way by looking at the increased outward K+ currents elicited in extracellular solutions in which K+ was replaced by Cs+. 4. The effects of TRH were mimicked by direct pharmacological activation of protein kinase C (PKC) with beta-phorbol 12-myristate, 13-acetate (PMA). The TRH-induced effects were antagonized by GF109203X, a highly specific inhibitor of PKC that also abolished the PMA-dependent regulation of the channels. 5. It is concluded that a PKC-dependent pathway links G protein-coupled receptors that activate phospholipase C to modulation of HERG channel gating. This provides a mechanism for the physiological regulation of cardiac function by phospholipase C-activating receptors, and for modulation of adenohypophysial neurosecretion in response to TRH.

A G protein beta gamma dimer-mediated pathway contributes to mitogen-activated protein kinase activation by thyrotropin-releasing hormone receptors in transfected COS-7 cells.

Activation of mitogen-activated protein kinase (MAPK) is induced by adding thyrotropin-releasing hormone (TRH) to COS-7 cells cotransfected with TRH receptors and an epitope-tagged MAPK. Long term treatment of the cells with pertussis toxin has no effect on TRH-induced MAPK activation. Incubation of the cells with the protein kinase C (PKC) inhibitor GF109203X causes an almost complete inhibition of MAPK activation by the PKC activator phorbol-12-myristate-13-acetate. In contrast, only approximately 50% of the TRH-induced MAPK activity is inhibited by GF109203X, indicating that activation of MAPK by TRH is only partially dependent on PKC. The inhibitory effect of GF109203X is additive with that of p21(N17ras), a dominant negative mutant of p21(ras) that exerts little effect on PKC-dependent MAPK activation by phorbol-12-myristate-13-acetate. The TRH-induced activation of MAPK also is inhibited partially by overexpression of transducin alpha subunits (alpha t), an agent known to sequester free G protein beta gamma dimers. However, the inhibitory potency of alpha t on TRH-induced activation is about half of that obtained in cells transfected with m2 muscarinic receptors, which activate MAPK exclusively through beta gamma dimers. The effect of alpha t is also additive with that of GF109203X but not with that of p21(N17ras). MAPK activation is not induced by the constitutively active form of G alpha q due to an inhibitory effect of its expression at a step downstream of that at which PKC-dependent and -independent routes to MAPK converge. Our results demonstrate that TRH receptors activate MAPK by a pathway only partially dependent on PKC activity. Furthermore, they indicate that beta gamma dimers of a pertussis and cholera toxin-insensitive G protein are involved in the PKC-independent fraction of the dual signaling route to MAPK initiated in the TRH receptor.