Sphingosine 1-phosphate restrains insulin-mediated keratinocyte proliferation via inhibition of Akt through the S1P2 receptor subtype.

The balance between keratinocyte proliferation and differentiation plays a decisive role for skin formation and development. Among the well-characterized biological mediators, insulin and sphingosine 1-phosphate (S1P) have been identified as major regulators of keratinocyte growth and differentiation. Insulin induces proliferation of keratinocytes, whereas S1P inhibits keratinocyte growth and initiates keratinocyte differentiation. However, it is not clear which S1P receptor subtype and downstream signaling pathways are involved in the antiproliferative action of S1P. In this study, we present evidence that S1P inhibits insulin-mediated keratinocyte growth via the activation of protein kinase C (PKC) followed by a subsequent dephosphorylation of Akt. The inhibition of insulin-mediated Akt activity by S1P is completely abolished in the presence of PKCdelta siRNA indicating that this isozyme is selectively potent at causing dephosphorylation of Akt and modifying keratinocyte proliferation. Further experiments by downregulation of S1P receptor subtypes and the use of specific receptor agonists/antagonists clearly indicated that the S1P(2) receptor is dominantly involved in the S1P-induced dephosphorylation of Akt and keratinocyte growth arrest. This is of great clinical interest, as the immunomodulator FTY720, after being phosphorylated by sphingosine kinase, activates all of the five S1P receptors except S1P(2) and therefore fails to inhibit keratinocyte proliferation.

Detection of subpicomolar concentrations of human matrix metalloproteinase-2 by an optical biosensor.

We describe in this paper the development of a one-step sandwich assay for the highly sensitive and fast detection of human matrix metalloproteinase (MMP)-2 (EC 3.4.24.24), using surface plasmon resonance (SPR). For the assay, two ligands were selected: monoclonal anti-MMP-2 antibody Ab-2 and the tissue inhibitor of metalloproteinases (TIMP)-2. They were chosen on the basis of (1) their affinities to MMP-2, (2) the efficiency of immobilization to the sensor chip, (3) the efficiency of adsorption to colloidal gold, and (4) the stability of these protein-coated gold particles. The assay included mixing of MMP-2 with antibody Ab-2 adsorbed to colloidal gold with a diameter of about 20nm and injection into the flowcell of the SPR instrument containing immobilized TIMP-2. By using colloidal gold particles an amplification factor of 114 and a detection limit of 0.5pM for MMP-2 were obtained. The precision of the assay was high even at low analyte concentrations, the standard deviation being 8.3% for five determinations of 1pM MMP-2. No significant binding was observed with the structurally related MMP-9. The assay is far more sensitive and faster than commonly used methods for MMP-2 detection. As TIMP-bound MMP-2 is not detected by this method, the assay can be applied for measuring free MMP-2, reflecting the imbalance of free and inhibitor-bound enzyme in various pathological situations.