Aquaporin-3 (AQP3) plays an important role in maintaining the normal water content of the skin. Previously, we revealed that the expression of cutaneous AQP3 increased following oral administration of Gypsum fibrosum (main component: CaSO 4 ) to mice. The purpose of this study is to elucidate the mechanism by which Gypsum fibrosum increases the expression of cutaneous AQP3 in a keratinocyte cell line. Gypsum fibrosum or CaSO 4 was added to keratinocytes, and the expression level of AQP3, the Ca concentration, the activity of protein kinase C (PKC), and the degrees of phosphorylation of both extracellular signal-regulated kinase (ERK) and cAMP response element binding protein (CREB) were measured. The mRNA and protein expression levels of AQP3 increased significantly 6 h-post addition of Gypsum fibrosum. In keratinocytes treated with Gypsum fibrosum, increases in the concentration of intracellular Ca, PKC activity, and the phosphorylation of ERK and CREB were observed. Pre-treatment with GF109203X, a PKC inhibitor, suppressed the mRNA expression levels of AQP3. Similarly to treatment with Gypsum fibrosum, the addition of CaSO 4 led to the same observations in keratinocytes. It is hypothesized that Gypsum fibrosum causes an increase in the intracellular Ca concentration, PKC activity, and the phosphorylation levels of ERK and CREB, resulting in increased AQP3 expression in keratinocytes. In addition, it is possible that the effect of Gypsum fibrosum is attributable to CaSO 4 , based on the results demonstrating that the mechanisms of action of Gypsum fibrosum and CaSO 4 were nearly identical.
Aquaporins (AQPs) are water channels found widely in bacteria, plants, animals, and humans. There are presently 13 known types of aquaporins in humans (AQP0 to AQP12), which are expressed in various organs. 1) In the kidney, several members of the AQP family are expressed, which are responsible for regulating water volume in the body. Especially in the renal collecting duct, AQP2, which is expressed on the luminal membranes of principal cells, and AQP3 and AQP4, which are expressed on the basolateral membranes of principal cells, are involved in urine concentration by inducing the re-absorption of water from the luminal side to the basolateral side.2) The capacity for water re-absorption in the renal collecting duct is thought to determine urine volume. Because either protein mutations or a decreased expression of AQP2 is found in patients with nephrogenic diabetes insipidus who present with marked polyuria, AQP2 is considered to play the most important role in water re-absorption in the renal collecting duct. 3,4) Enhanced expression of renal AQP2 has been reported when polyuria occurs in streptozotocin (STZ)-induced type I diabetes model mice. 5,6) In addition, we previously found that the level of renal AQP2 expression increases along with the pathological progression of type II diabetes mellitus using KKAy mice, the animal model of type II diabetes. 7) Although this increased AQP2 expression in the kidneys has been regarded as a possible compensatory mechanism to alleviate dehydration in polyuria, the physiological significance of the increase in renal AQP2 expression in diabetes remains unknown. Recently, a method to generate a mouse model of nephrogenic diabetes insipidus by administering lithium carbonate (Li 2 CO 3 ) was reported. 8,9) Almost all of the Li 2 CO 3 absorbed into the body is eliminated unchanged via glomerular filtration. Lithium is incorporated into principal cells of the collecting tubules via epithelial sodium channels that are dominantly expressed on the collecting tubules.10) The incorporated lithium decreases cAMP by suppressing the activity of adenylate cyclase (AC), resulting in the inhibition of pathways for both AQP2 and AQP3 transcription and membrane transfer. 8,9,11) Using Li 2 CO 3 , we constructed an experimental system to decrease only the expression levels of AQPs, with no change in urinary osmotic pressure, to investigate the relationship between urine volume and the expression levels of AQPs. In this study, we developed this experimental system by administering Li 2 CO 3 to STZ-induced type I diabetes model mice. MATERIALS AND METHODSMaterials 2-Mercaptoethanol, bromophenol blue, Folin and Ciocalteu's phenol reagent, polyoxyethylene (20) sorbitan monolaurate (Tween 20), sodium dodecyl sulphate (SDS), and 2-amino-2-hydroxymethyl-1,3-propanediol (Tris) were purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). Anti-rat aquaporin 1, anti-rat aquaporin 2, anti-rat aquaporin 3, and anti-rat aquaporin 4 antibodies were purchased from Alomone Labs, Ltd. (Jerusalem, ...
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