Transcriptional and Post-Transcriptional Regulation of the CTNS Gene

PROGRESS REPORT

Date: Rome, September 2nd, 2007

PROJECT: Transcriptional and post-transcriptional regulation of the CTNS gene
PRINCIPAL INVESTIGATOR: Francesco Emma, MD
CO-INVESTIGATOR: Elena Levtchenko, MD and Anna Taranta, PhD
FIRST SIX MONTH PERIOD: March 1st – September 1st, 2007

The main goal of this project is to characterize a new cystinosin isoform (cystinosin-LKG) resulting from an alternative splicing of the exon 12 of the CTNS gene. 
We have began the experimental work that was proposed, as briefly summarized below.  We have also concluded with the support of the Cystinosis Research Foundation some complementary experiments that have been included in a manuscript describing the cystinosin-LKG isoform, which is currently under review.  Support of the Cystinosis Research Foundation is acknowledged in the manuscript.  Again, we are very grateful for the support and hope to produce interesting results in the very next future.

Specific aim #1: Characterization of the cystinosin-LKG isoform
Proximal tubular cells (PTC) of two cystinosis patients and two healthy subjects have been cultured succesfully and have subsequently been conditionally immortalized with SV-40 T antigen and subcloned. The SV-40 T protein is not expressed at 10 days culturing at 37C. The conditionally immortalized cells express the proximal tubule markers aquaporin-1, p-glycoprotein and CD26 as shown by immunoblot techniques (fig 1) indicating their proximal tubular origin.

The conditionally immortalized cells of both cystinosis patients and healthy controls are transiently transfected using Lipofectamin 2000 with vectors containing two isoforms of CTNS, called CTNS and CTNS-LKG, both with a RED fusion protein for localisation studies. Subsequently, transfected cells are incubated with antibodies raised again CTNS-LKG and visualized using conjugate go-a-rb-Alexa 488 (green). Cells are analyzed using immunofluorescence (fig 2). Both vectors are expressed in a intracellular vesicular pattern indicating succesful transfection. Only isoform CTNS-LKG perfectly co-localizes with CTNS-LKG antibodies.

Specific aim #2: Analysis of the relative expression of the cystinosin isoforms
The expression of the two cystinosin isoforms has been analyzed in different human tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, speen, thymus, prostate, testis, ovary, small intestine, colon and peripheral leukocytes).
Cystinosin-LKG expression is tissue-specific, with the highest expression in the testis. This result may have important implications in explaining male hypogonadism in NC.
In addition, we have began analyzing the differential expression of the 2 isoforms under different conditions, including cystine deprivation and various forms of oxidative stress.  So far, we have not detected significant changes in the relative expression of the two isoforms under the conditions that have been tested.

Specific aim #3: Characterization of the cystinosin promoter
Constructs containing partial regions of the CTNS promoter have been prepared (from –769 bp, -348 bp, -316 bp, -254 bp, -81 bp to +1 bp) and cloned into the luciferase pGL4 vector.  HK2 cells have been co-transfected with the pGL4 vector and the control phRL-TK vector that mediates the expression of the Renilla luciferase for normalization.
The activity of the promotor has been verified, reproducing the data that have been published by Phornphutkul et al.  We are now in the process of testing the promoter under different stimuli and are also expanding the range of experiments that have been initially proposed including RNA stability experiments.