Scleroderma Australia

Title: Identification of novel markers and molecular targets in pathological
fibrosis in scleroderma skin lesions

Chief Investigators : Pravin Hissaria, Yeesim Khew-Goodall, Susanna Proudman
Division of Human Immunology, Istitute of Medical and Veterinary Science/SA Pathology, Adelaide SA 5000

We were successful in receiving an NHMRC project grant based upon the pilot work undertaken with the research money obtained from the Scleroderma Australia grant in 2009.
The progress report on the NHMRC work is detailed below.  

Application ID:  626945  CIA Y Khew-Goodall, CIB G Goodall, CIC Susanna Proudman,
CID P Hissaria
Project Grant:  Identification of microRNAs in Scleroderma
Commencement date: 2010

Aims:
1.     To identify targets of the microRNAs that are altered by TGF?-induced transdifferentiation of     dermal fibroblast to myofibroblast.
2.    To test the hypothesis that microRNAs involved in the process of TGF?-induced fibrosis in vitro are altered in scleroderma skin fibrosis.
3.    To test the hypothesis that enforced expression of pre-miRs or anti-miRs can block or reverse the TGF?-induced transdifferentiation.
4.    To test the hypothesis that TGF?-induced EMT of epidermal keratinocytes leads to alterations in microRNA expression

Aim 1: In this study, we have focused on the miR-29 family and miR-424 microRNAs as these were found to be consistently downregulated or upregulated, respectively, in TGF?-induced transdifferentiation of primary human dermal fibroblasts. We identified a number of collagens (5A1, 4A1, 4A2, 7A1, 1A1, 16A1 & 5A2) as well as 2 members of the lysyl oxidase family of collagen bundling enzymes (LOXL2, LOXL4) as targets of miR-29. We have generated reporter constructs with wt and mutated miR-29 binding sites in the 3'UTRs of LOXL2 & LOXL4 and are currently verifying that they are direct targets of miR-29 as these targets have not previously been identified. We have identified SEMA7A as an indirect target of miR-424 that is of special interest to Ssc as it has previously been shown to be involved in Ssc.
Aim 2: We have collected involved and uninvolved (in the case of limited disease) skin biopsies from 14 different Ssc patients, as well as normal skin from abdominoplasty as controls and have begun microdissection of the dermis and epidermis for multiplex qPCR to identify differentially expressed microRNAs between normal and Ssc skins. Initial qPCR studies have revealed a number of new microRNAs whose expression may be related to Ssc. We are currently verifying these observations.
Aim 3: We have generated constructs (lentiviral-based) to stably express pre-miR-29 and anti-miR-424 and transduced these into primary dermal fibroblasts. We have tested the miR-29 construct and shown that it is functional and can block TGF?-induced upregulation of collagen and LOXL. Work on miR-424 in progress have identified a number of downstream mediators of fibrosis. We are currently verifying if these changes can be blocked using anti-miR424 strategies.
Aim 4:  Work is continuing to further characterize this process and to identify other microRNAs whose expression may be altered by TGF? treatment.