Disruption of Retinoic Acid Receptor Alpha Reveals the Growth Promoter Face of Retinoic Acid

Retinoic acid (RA), the bioactive derivative of Vitamin A, by epigenetically controlling transcription through the RA-receptors (RARs), exerts a potent antiproliferative effect on human cells. However, a number of studies show that RA can also promote cell survival and growth. In the course of one of our studies we observed that disruption of RA-receptor alpha, RARalpha, abrogates the RA-mediated growth-inhibitory effects and unmasks the growth-promoting face of RA (Ren et al., Mol. Cell. Biol., 2005, 25:10591). The objective of this study was to investigate whether RA can differentially govern cell growth, in the presence and absence of RARalpha, through differential regulation of the "rheostat" comprising ceramide (CER), the sphingolipid with growth-inhibitory activity, and sphingosine-1-phosphate (S1P), the sphingolipid with prosurvival activity.We found that functional inhibition of endogenous RARalpha in breast cancer cells by using either RARalpha specific antagonists or a dominant negative RARalpha mutant hampers on one hand the RA-induced upregulation of neutral sphingomyelinase (nSMase)-mediated CER synthesis, and on the other hand the RA-induced downregulation of sphingosine kinase 1, SK1, pivotal for S1P synthesis. In association with RA inability to regulate the sphingolipid rheostat, cells not only survive, but also grow more in response to RA both in vitro and in vivo. By combining genetic, pharmacological and biochemical approaches, we mechanistically demonstrated that RA-induced growth is, at least in part, due to non-RAR-mediated activation of the SK1-S1P signaling.In the presence of functional RARalpha, RA inhibits cell growth by concertedly, and inversely, modulating the CER and S1P synthetic pathways. In the absence of a functional RARalpha, RA-in a non-RAR-mediated fashion-promotes cell growth by activating the prosurvival S1P signaling. These two distinct, yet integrated processes apparently concur to the growth-promoter effects of RA

Acute diverticulitis management: evolving trends among Italian surgeons. A survey of the Italian Society of Colorectal Surgery (SICCR)

Acute diverticulitis (AD) is associated with relevant morbidity/mortality and is increasing worldwide, thus becoming a major issue for national health systems. AD may be challenging, as clinical relevance varies widely, ranging from asymptomatic picture to life-threatening conditions, with continuously evolving diagnostic tools, classifications, and management. A 33-item-questionnaire was administered to residents and surgeons to analyze the actual clinical practice and to verify the real spread of recent recommendations, also by stratifying surgeons by experience. CT-scan remains the mainstay of AD assessment, including cases presenting with recurrent mild episodes or women of child-bearing age. Outpatient management of mild AD is slowly gaining acceptance. A conservative management is preferred in non-severe cases with extradigestive air or small/non-radiologically drainable abscesses. In severe cases, a laparoscopic approach is preferred, with a non-negligible number of surgeons confident in performing emergency complex procedures. Surgeons are seemingly aware of several options during emergency surgery for AD, since the rate of Hartmann procedures does not exceed 50% in most environments and damage control surgery is spreading in life-threatening cases. Quality of life and history of complicated AD are the main indications for delayed colectomy, which is mostly performed avoiding the proximal vessel ligation, mobilizing the splenic flexure and performing a colorectal anastomosis. ICG is spreading to check anastomotic stumps' vascularization. Differences between the two experience groups were found about the type of investigation to exclude colon cancer (considering the experience only in terms of number of colectomies performed), the size of the peritoneal abscess to be drained, practice of damage control surgery and the attitude towards colovesical fistula

Impaired Retinoic Acid (RA) Signal Leads to RARβ2 Epigenetic Silencing and RA Resistance

Resistance to the growth-inhibitory action of retinoic acid (RA), the bioactive derivative of vitamin A, is common in human tumors. One form of RA resistance has been associated with silencing and hypermethylation of the retinoic acid receptor β2 gene (RARβ2), an RA-regulated tumor suppressor gene. The presence of an epigenetically silent RARβ2 correlates with lack of the RA receptor α (RARα). Normally, RARα regulates RARβ2 transcription by mediating dynamic changes of RARβ2 chromatin in the presence and absence of RA. Here we show that interfering with RA signal through RARα (which was achieved by use of a dominant-negative RARα, by downregulation of RARα by RNA interference, and by use of RARα antagonists) induces an exacerbation of the repressed chromatin status of RARβ2 and leads to RARβ2 transcriptional silencing. Further, we demonstrate that RARβ2 silencing causes resistance to the growth-inhibitory effect of RA. Apparently, RARβ2 silencing can also occur in the absence of DNA methylation. Conversely, we demonstrate that restoration of RA signal at a silent RARβ2 through RARα leads to RARβ2 reactivation. This report provides proof of principle that RARβ2 silencing and RA resistance are consequent to an impaired integration of RA signal at RARβ2 chromatin

RARα antagonists counteract both RA-induced growth inhibition and nSMase-mediated CER synthesis.

<p>A) RO415253 (RO) (left) and ER50891 (ER) (right) rescued T47D cells from RA-induced growth-inhibition. B) Both RO and ER counteracted the RA-induced CER synthesis. C) Both RO and ER significantly counteracted the transcription of <i>nSMase</i> as well as the transcription of the control downstream RARα direct target <i>RARβ2</i>.</p

RA fails to induce nSMase-mediated CER synthesis in cells with functional RARα inhibition.

<p>A) Scheme showing the metabolic pathways leading to the synthesis of both CER and S1P, two bioactive sphingolipids known for exerting opposite effects on proliferation. B) [³H] palmitate or [³H] sphingosine labelling, in the presence and absence of RA, showing that RA induces CER accumulation through a sphingomyelinase pathway (fluorographic pattern of CER and sphingomyelin (SM), respectively, in the upper inserts). C) Quantitative analysis of transcription and activity for the different enzymes leading to CER synthesis in both LXC5 cells and DNC8 cells clearly indicates that RA induces CER through the nSMase pathway (middle) and not the SPT (top) and aSMase (bottom) pathways. D) The nSMase inhibitor GW4869 significantly counteracted both RA-induced growth inhibition (left) and CER accumulation (right).</p

SK1-S1P signaling: a candidate growth promoting mechanism of non-RAR-mediated RA action.

<p>A) Transient exogenous expression of SK1 in DNC8 cells leads to upregulation of cyclin D1 transcription relative to cells expressing the cognate empty vector (left). Conversely, transient exogenous expression of a dominant negative SK mutant in DNC8 cells negatively affects RA-induced cyclin D1 transcription (right). B) RA-induced DNC8 proliferation is significantly decreased by treatment with a SK inhibitor. C) RA upregulates the S1P level (spots in the upper insert) in DNC8 cells. D) Scheme showing that RA action mediated through RARα results in upregulation of nSMase-generated CER sythesis, concomitant with downregulation of SK1 transcription/activity. These concerted antiproliferative metabolic changes concur to inhibit cell proliferation. Consequent to an impaired RA-RARα signaling, these concerted antiproliferative metabolic changes do not occur, thus cells survive in the presence of RA. Moreover, RA, through alternate, non-RAR (genomic or non-genomic) target(s), activates pro-survival signaling pathways, including the SK signaling pathway, thus leading to the expansion of the RA-resistant cell pool.</p

Evidence that RA fails to regulate in an opposite fashion CER synthesis and SK activity in cells lacking endogenous RARα.

<p>A) Lack of endogenous RARα signaling (left) is demonstrated by lack of RA-induced transcription of the RARα target <i>RARβ2</i> (right). B) MDA-MB-231 are modestly, but significantly, growth-promoted by RA. C) RA fails to induce <i>nSMase</i> transcription (left), CER synthesis (middle), <i>SK1</i> transcription downregulation (middle), and decrease of SK activity (right) in MDA-MB-231 cells. D) RA-induced MDA-MB-231 proliferation is significantly decreased by treatment with a SK inhibitor. E) Fenretinide can effectively induce both CER synthesis (left) and apoptosis (right) in MDA-MB-231 cells.</p

Fenretinide can induce CER also in cells without a functional RARα.

<p>A) DGK analysis of CER (autoradiography of CER species in the upper inserts) showing that RA induces CER only in cells with a functional RARα signaling (left), while fenretinide, a retinoid whose action is RAR-independent, can induce CER also in cells without a functional RARα signaling (right). B) Fenretinide-induced CER accumulation has an antiproliferative and proapoptotic effect, as shown by cell proliferation assay (left) and the presence of cleaved PARP (right), respectively.</p