Novel role of parathyroid hormone-related protein in the pathophysiology of the diabetic kidney: evidence from experimental and human diabetic nephropathy.

Parathyroid hormone-related protein (PTHrP) and its receptor type 1 (PTH1R) are extensively expressed in the kidney, where they are able to modulate renal function. Renal PTHrP is known to be overexpressed in acute renal injury. Recently, we hypothesized that PTHrP involvement in the mechanisms of renal injury might not be limited to conditions with predominant damage of the renal tubulointerstitium and might be extended to glomerular diseases, such as diabetic nephropathy (DN). In experimental DN, the overexpression of both PTHrP and the PTH1R contributes to the development of renal hypertrophy as well as proteinuria. More recent data have shown, for the first time, that PTHrP is upregulated in the kidney from patients with DN. Collectively, animal and human studies have shown that PTHrP acts as an important mediator of diabetic renal cell hypertrophy by a mechanism which involves the modulation of cell cycle regulatory proteins and TGF- ß 1. Furthermore, angiotensin II (Ang II), a critical factor in the progression of renal injury, appears to be responsible for PTHrP upregulation in these conditions. These findings provide novel insights into the well-known protective effects of Ang II antagonists in renal diseases, paving the way for new therapeutic approaches.

Gene expression profiling of subcutaneous adipose tissue in morbid obesity using a focused microarray: distinct expression of cell-cycle- and differentiation-related genes.

BACKGROUND: Obesity results from an imbalance between food intake and energy expenditure, which leads to an excess of adipose tissue. The excess of adipose tissue and adipocyte dysfunction associated with obesity are linked to the abnormal regulation of adipogenesis. The objective of this study was to analyze the expression profile of cell-cycle- and lipid-metabolism-related genes of adipose tissue in morbid obesity. METHODS: We used a custom-made focused cDNA microarray to determine the adipose tissue mRNA expression profile. Gene expression of subcutaneous abdominal fat samples from 15 morbidly obese women was compared with subcutaneous fat samples from 10 nonobese control patients. The findings were validated in an independent population of 31 obese women and 9 obese men and in an animal model of obesity (Lepob/ob mice) by real-time RT-PCR. RESULTS: Microarray analysis revealed that transcription factors that regulate the first stages of adipocyte differentiation, such as CCAAT/enhancer binding protein beta (C/EBPß) and JUN, were upregulated in the adipose tissues of morbidly obese patients. The expression of peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor which controls lipid metabolism and the final steps of preadipocyte conversion into mature adipocytes, was downregulated. The expression of three cyclin-dependent kinase inhibitors that regulate clonal expansion and postmitotic growth arrest during adipocyte differentiation was also altered in obese subjects: p18 and p27 were downregulated, and p21 was upregulated. Angiopoietin-like 4 (ANGPTL4), which regulates angiogenesis, lipid and glucose metabolism and it is know to increase dramatically in the early stages of adipocyte differentiation, was upregulated. The expression of C/EBPß, p18, p21, JUN, and ANGPTL4 presented similar alterations in subcutaneous adipose tissue of Lepob/ob mice. CONCLUSIONS: Our microarray gene profiling study revealed that the expression of genes involved in adipogenesis is profoundly altered in the subcutaneous adipose tissue of morbidly obese subjects. This expression pattern is consistent with an immature adipocyte phenotype that could reflect the expansion of the adipose tissue during obesity.

Induction of the endoplasmic reticulum stress protein GADD153/CHOP by capsaicin in prostate PC-3 cells: a microarray study.

The effect of capsaicin, main pungent ingredient of hot chilli peppers, in the gene expression profile of human prostate PC-3 cancer cells has been analyzed using a microarray approach. We identified 10 genes that were down-regulated and five genes that were induced upon capsaicin treatment. The data obtained from microarray analysis were then validated using quantitative real-time PCR assays and Western blot analysis. The most remarkable change was the up-regulation of GADD153/CHOP, an endoplasmic reticulum stress-regulated gene. Activation of GADD153/CHOP protein was corroborated by immunofluorescence and Western blot. We then tested the contribution of GADD153/CHOP to protection against capsaicin-induced cell death using RNA interference. Blockage of GADD153/CHOP expression by small interfering RNA, significantly reduced capsaicin-induced cell death in PC-3 cells. Taken together, these results suggested that capsaicin induces the antiproliferative effect through a mechanism facilitated by ER stress in prostate PC-3 cells.

Spisulosine (ES-285) induces prostate tumor PC-3 and LNCaP cell death by de novo synthesis of ceramide and PKCzeta activation.

During the past decades, intense attention has been focused on the anti-tumor properties of marine compounds which some of them have been revealed as potent apoptotic inducers. In the present work, we studied the mechanism of action of a new compound, Spisulosine (ES-285), isolated from the sea mollusc Spisula polynyma, in the prostate tumor PC-3 and LNCaP cell lines. Spisulosine inhibited cell proliferation with an IC50 of 1 microM in both cell lines, although it was more effective in the androgen-independent PC-3 cells. The anti-proliferative effect induced by Spisulosine in prostate cells was independent of peroxisome proliferator activated receptor gamma (PPARgamma) and phosphatidylinositol 3-kinase/(PI3K/Akt), Jun N-terminal kinase (JNK), p38 or classical protein kinase C (PKCs) pathways, as it was inferred from the results obtained with specific inhibitors of these routes. However, Spisulosine treatment of prostate cells induced an increase in the intracellular ceramide levels, that was totally blocked by the ceramide synthase inhibitor Fumonisin B1, indicating that the ceramide accumulation came from the de novo biosynthesis. Spisulosine also induced in both PC-3 and LNCaP cells, an activation of the atypical PKC isoform, PKCzeta, which is one of the target proteins of ceramide. These results indicate that the marine compound Spisulosine inhibits the growth of the prostate PC-3 and LNCaP cells through intracellular ceramide accumulation and PKCzeta activation.

Apoptosis induced by capsaicin in prostate PC-3 cells involves ceramide accumulation, neutral sphingomyelinase, and JNK activation.

Numerous studies have recently focused on the anticarcinogenic, antimutagenic, or chemopreventive activities of the main pungent component of red pepper, capsaicin (N-vanillyl-8-methyl-1-nonenamide). We have previously shown that, in the androgen-independent prostate cancer PC-3 cells, capsaicin inhibits cell growth and induces apoptosis through reactive oxygen species (ROS) generation [Apoptosis 11 (2006) 89-99]. In the present study, we investigated the signaling pathways involved in the antiproliferative effect of capsaicin. Here, we report that capsaicin apoptotic effect was mediated by ceramide generation which occurred by sphingomyelin hydrolysis. Using siRNA, we demonstrated that N-SMase expression is required for the effect of capsaicin on prostate cell viability. We then investigated the role of MAP kinase cascades, extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, in the antiproliferative effect of capsaicin, and we confirmed that capsaicin could activate ERK and JNK but not p38 MAPK. Pharmacological inhibition of JNK kinase, as well as inhibition of ROS by the reducing agent N-acetylcysteine, prevented ceramide accumulation and capsaicin-induced cell death. However, inhibition of ceramide accumulation by the SMase inhibitor D609 did not modify JNK activation. These data reveal JNK as an upstream regulator of ceramide production. Capsaicin-promoted activation of ERK was prevented with all the inhibitors tested. We conclude that capsaicin induces apoptosis in PC-3 cells via ROS generation, JNK activation, ceramide accumulation, and second, ERK activation.

Expression of the transient receptor potential vanilloid 1 (TRPV1) in LNCaP and PC-3 prostate cancer cells and in human prostate tissue.

Vanilloid receptor subtype-1 (TRPV1), the founding member of the vanilloid receptor-like transient receptor potential channel family, is a non-selective cation channel that responds to noxious stimuli such as low pH, painful heat and irritants. In the present study, we show, as means of reverse transcriptase-polymerase chain reaction and Western blot analysis, that the vanilloid TRPV1 receptor is expressed in the prostate epithelial cell lines PC-3 and LNCaP as well as in human prostate tissue. The kinetic parameters inferred from [(125)I]-resiniferatoxin binding were in concordance with data of TRPV1 receptors expressed in other tissues. The contribution of the endogenously expressed TRPV1 channel to intracellular calcium concentration increase in the prostate cells was studied by measuring changes in Fura-2 fluorescence by fluorescence microscopy. Addition of capsaicin, (R)-methanandamide and resiniferatoxin to prostate cells induced a dose-dependent increase in the intracellular calcium concentration that was reversed by the vanilloid TRPV1 receptor antagonist capsazepine. These results indicate that the vanilloid TRPV1 receptor is expressed and functionally active in human prostate cells.