Chronic low-dose isotretinoin treatment limits renal damage in subtotally nephrectomized rats.

Retinoids are anti-proliferative and anti-inflammatory compounds. We had previously shown that retinoids alleviate kidney damage in acute models of renal disease. We now examined whether retinoids are also effective in a chronic renal ablation model. Subtotally nephrectomized rats (SNx; two-third ablation) were compared to sham-operated controls (sham). SNx rats were administered either 10 mg/kg b.w. (low dose, LD) or 40 mg/kg b.w. (high dose, HD) isotretinoin or vehicle (n = 10 per group). The experiment was terminated after 16 weeks. Systolic blood pressure was significantly higher after SNx compared to sham but lower in SNx with LD isotretinoin (vs. SNx + vehicle). Compared to SNx + vehicle, SNx + LD isotretinoin had lower glomerular cell numbers, less glomerular hypertrophy and sclerosis, and less interstitial expansion. Morphological improvement in SNx + LD isotretinoin was accompanied by improvement in creatinine clearance and reduced urinary albumin excretion. In contrast, HD isotretinoin caused aggravation of renal damage with fibrinoid necroses of vessels and elevated urinary albumin excretion despite lower blood pressure. The dichotomous effects of isotretinoin are at least in part due to time- and dose-dependent alterations of transforming growth factor beta1 and collagen IV gene expression as also suggested by cell-culture studies in vascular smooth muscle cells. In addition, isotretinoin affected the systemic and the renal renin-angiotensin system (which was further analyzed in a model of angiotensin II infusion of the rat). Isotretinoin failed to cumulate at LD but cumulated at HD in SNx. We conclude that LD isotretinoin attenuates progressive renal damage, whereas HD isotretinoin cumulates and aggravates renal damage independent of blood pressure reduction.

Neurabin II mediates doublecortin-dephosphorylation on actin filaments.

Mutations in the human Doublecortin (DCX) gene cause X-linked lissencephaly, a neuronal migration disorder. DCX binds to microtubules and actin filaments. Association of Dcx with F-actin is regulated by site-specific phosphorylation and by neurabin II, an F-actin binding protein that also binds to Dcx. We show here that neurabin II mediates dephosphorylation of Dcx by protein phosphatase 1 (PP1). Furthermore, overexpression of PP1 reduces Dcx phosphorylation and decreases Dcx binding to F-actin. By contrast, abolishing PP1 binding to neurabin II maintains phosphorylation levels of Dcx, leading to a retention of Dcx at F-actin. We suggest that a dynamic regulation of Dcx mediated by neurabin II regulates the translocation of Dcx from F-actin to microtubules and vice versa.

Site-specific dephosphorylation of doublecortin (DCX) by protein phosphatase 1 (PP1).

Mutations in doublecortin (DCX) cause X-linked lissencephaly ("smooth brain") and double cortex syndrome in humans. DCX is highly phosphorylated in migrating neurons. Here, we demonstrate that dephosphorylation of specific sites phosphorylated by JNK is mediated by Neurabin II, which recruits the phosphatase PP1. During cortical development, the expression pattern of PP1 is widespread, while the expression of DCX and Neurabin II is dynamic, and they are coexpressed in migrating neurons. In vitro, DCX is site-specific dephosphorylated by PP1 without the presence of Neurabin II, this dephosphorylation requires an intact RVXF motif in DCX. Overexpression of the coiled-coil domain of Neurabin II, which is sufficient for interacting with DCX and recruiting the endogenous Neurabin II with PP1, induced dephosphorylation of DCX on one of the JNK-phosphorylated sites. We hypothesize that the transient recruitment of DCX to different scaffold proteins, JIP-1/2, which will regulate its phosphorylation by JNK, and Neurabin II, which will regulate its dephosphorylation by PP1, plays an important role in normal neuronal migration.

Doublecortin association with actin filaments is regulated by neurabin II.

Mutations in the human Doublecortin (DCX) gene cause X-linked lissencephaly, a neuronal migration disorder affecting the neocortex and characterized by mental retardation and epilepsy. Because dynamic cellular asymmetries such as those seen in cell migration critically depend on a cooperation between the microtubule and actin cytoskeletal filament systems, we investigated whether Dcx, a microtubule-associated protein, is engaged in cytoskeletal cross-talk. We now demonstrate that Dcx co-sediments with actin filaments (F-actin), and using light and electron microscopy and spin down assays, we show that Dcx induces bundling and cross-linking of microtubules and F-actin in vitro. It has recently been shown that binding of Dcx to microtubules is negatively regulated by phosphorylation of the Dcx at Ser-47 or Ser-297. Although the phosphomimetic green fluorescent protein (GFP)-Dcx(S47E) transfected into COS-7 cells had a reduced affinity for microtubules, we found that pseudophosphorylation was not sufficient to cause Dcx to bind to F-actin. When cells were co-transfected with neurabin II, a protein that binds F-actin as well as Dcx, GFP-Dcx and to an even greater extent GFP-Dcx(S47E) became predominantly associated with filamentous actin. Thus Dcx phosphorylation and neurabin II combinatorially enhance Dcx binding to F-actin. Our findings raise the possibility that Dcx acts as a molecular link between microtubule and actin cytoskeletal filaments that is regulated by phosphorylation and neurabin II.

The renal retinoid system: time-dependent activation in experimental glomerulonephritis.

Retinoids reduce renal damage in rat experimental glomerulonephritis. It is unknown, however, how local and systemic retinoid pathways respond to renal injury. We used a rat model of artificially induced acute anti-Thy1.1-nephritis (THY-GN). We examined the extrarenal and glomerular expression of the retinol (RoDH) and retinal (RalDH) dehydrogenases 1 and 2 as well as the expression of the retinoic acid (RAR) and retinoid X (RXR) receptor subtypes alpha, beta, and gamma. Furthermore, we investigated serum and glomerular retinoid concentration patterns. On days 3, 7, and 14, we compared nonnephritic rats (control group; CON) to THY-GN rats with respect to systolic blood pressure and glomerular cell count per cross section. Systolic blood pressure and glomerular cell count were significantly higher in THY-GN rats on days 7 and 14 (P < 0.001). We found a 60% reduction in expression levels for retinoid receptors and dehydrogenases in nephritic glomeruli on day 3, but a threefold increase on day 7 (P < 0.001 vs. CON). The same applies to RAR alpha protein. Hepatic expression of retinoid receptors was not influenced. On day 14, glomerular expression levels for retinoid receptors and retinoid-metabolizing enzymes had returned to a normal level, glomerular cell count being still increased. Administering 13-cis retinoic acid (isotretinoin) lowered blood pressure and glomerular cell count in nephritic rats but failed to influence the glomerular expression of retinoid receptors or retinoid-metabolizing enzymes. Our data document a stimulation of glomerular retinoid-synthesizing enzymes and expression of retinoid receptors in the early repair phase of THY-GN, suggesting activation of this system in acute renal disease.

Identification of neurabin II as a novel doublecortin interacting protein.

The neuronal migration protein doublecortin (DCX) that associates with microtubules through a tandem DCX repeat, is required for the development of the complex architecture of the human cerebral cortex. Using a yeast two-hybrid screen with Dcx as bait, we have isolated neurabin II/spinophilin, an F-actin binding protein known to play a role in dendritic spine formation. The coiled-coil domain of neurabin II binds to a DCX region encompassing the C-terminal portion of the second DCX repeat and the N-terminal portion of the Ser/Pro-rich domain. Immunoprecipitation experiments with brain extracts show that neurabin II and Dcx interact in vivo. Several Dcx constructs that mimic human DCX mutant alleles failed to interact with neurabin II. Since Dcx and neurabin II colocalized in the developing and adult brain, a neurabin II-DCX heterodimer may be involved in neuronal migration and dendritic spine formation.

Differentiation and apoptosis in human immortalized sebocytes.

Increased cell volume, accumulation of lipid droplets in the cytoplasm, and nuclear degeneration are phenomena indicating terminal differentiation of human sebocytes followed by holocrine secretion and cell death. The molecular pathways of natural and induced sebocyte elimination are still unknown, however. In this study, SZ95 sebocytes were found to exhibit DNA fragmentation after a 6 h culture followed by increased lactate dehydrogenase release after 24 h, indicating cell damage. With the help of morphologic studies and using Oil Red detection of cellular lipids, cell enlargement, accumulation of lipid droplets in the cytoplasm, and nuclear fragmentation could be observed under treatment with arachidonic acid. Staurosporine, a potent inhibitor of phospholipid Ca2+-dependent protein kinase, increased externalized phosphatidylserine levels on SZ95 sebocytes, detected by annexin V/propidium iodide flow cytometry, as early as after 1 h, whereas dose-dependent reduction of bcl-2 mRNA and protein expression, enhanced DNA fragmentation, and increased caspase 3 levels, detected by caspase 3 inhibitor/propidium iodide flow cytometry, were found after 6 h of treatment. SZ95 sebocyte death was detected as early as after 6 h of SZ95 sebocyte treatment with high staurosporine concentrations (10(-6)-10(-5) M). 5Alpha-dihydrotestosterone (10(-8)-10(-5) M) did not affect externalized phosphatidylserine levels and DNA fragmentation in SZ95 sebocytes but slightly decreased lactate dehydrogenase cell release. Neither acitretin nor 13-cis retinoic acid (10(-8)-10(-5) M) affected externalized phosphatidylserine levels, DNA fragmentation, and lactate dehydrogenase cell release, despite the increased caspase 3 levels under treatment with 13-cis retinoic acid. The combined staurosporine and 13-cis retinoic acid treatment enhanced DNA fragmentation in SZ95 sebocytes to the same magnitude as in cells only treated with staurosporine. In conclusion, SZ95 sebocytes in vitro undergo apoptosis, which can be enhanced by the terminal differentiation inductor arachidonic acid or by staurosporine and leads to cell death. 5Alpha-dihydrotestosterone inhibits SZ95 sebocyte death without involving apoptotic pathways, and retinoids did not affect the programmed death of human sebocytes. The latter result fits well with the currently reported inability of normal skin cells to undergo apoptosis after treatment with retinoids, in contrast to their malignant counterparts.

High albumin levels restrict the kinetics of 13-cis retinoic acid uptake and intracellular isomerization to all-trans retinoic acid and inhibit its anti-proliferative effect on SZ95 sebocytes.

13-cis Retinoic acid is rapidly absorbed into cells and exerts its anti-proliferative effect on human sebocytes by specific isomerization to high levels of all-trans retinoic acid and binding the retinoic acid receptors. In this study, we have shown that bovine serum albumin, an extracellular binding protein for 13-cis retinoic acid, plays an important part in the uptake of 13-cis retinoic acid in human sebocytes, its intracellular isomerization to all-trans retinoic acid, and the induction of its anti-proliferative effect. The addition of highly concentrated bovine serum albumin (20 mg per ml) to the serum-free maintenance medium resulted in a rather controlled uptake of constant levels of 13-cis and all-trans retinoic acid into the cells over the 72 h of treatment. As a consequence, significantly reduced and delayed isomerization of 13-cis retinoic acid to all-trans retinoic acid was detected. In parallel experiments, the anti-proliferative activity of 13-cis retinoic acid on SZ95 sebocytes was abrogated by adding 20 mg bovine serum albumin per ml into the serum-free medium. These results indicate a critical function of serum albumin as retinoid-binding protein in reducing the concentration of active retinoids and restricting their biologic effects on human sebocytes.