2016 Fiscal Year Research-status Report
Determination of the functions of specific CD44 proteins in human skin and epidermis and their relation to skin diseases, cancer and autoimmunity
| Project/Area Number |
16K10137
|
|---|
| Research Institution | Kurume University |
|---|
Principal Investigator |
TEYE KWESI 久留米大学, 皮膚細胞生物学研究所, 研究員 (30599303)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
橋本 隆 久留米大学, 皮膚細胞生物学研究所, 教授 (20129597)
沼田 早苗 岩手医科大学, いわて東北メディカル・メガバンク機構 イノベーション推進・人材育成部門, 特命助教 (40599312)
石井 文人 久留米大学, 医学部, 准教授 (80330827)
|
|---|
| Project Period (FY) |
2016-04-01 – 2019-03-31
|
| Keywords | CD44 / Human Skin / Keratinocyte / Skin Cancer / Squamous cell carcinoma / Skin disease |
|---|
| Outline of Annual Research Achievements |
We previously identified up to 18 different CD44 transcripts in human epidermis. The functions of most of these transcripts are not well characterized and the purpose of this research is to determine the functions of CD44 proteins in human epidermis.
Traditionally, it is difficult to deliver gene for over-expression or down-regulation into human keratinocytes. We have determined the best ways to introduce foreign gene into human keratinocytes. We found that use of xfect transfection reagent and use of lentiviral delivery systems are the best ways to introduce a foreign gene into keratinocytes. We have now constructed vectors containing specific CD44 cDNA or siRNA to enable us introduce them into keratinocytes to study the effect of over expression or down regulations of specific CD44 molecules in human epidermis and skin.
We also studied the expression of CD44 variant expression in 2 skin autoimmune diseases, Pemphigus vulgaris (PV) and Bullous pemphigoid (BP). We did not find reduced expression of CD44 proteins in these diseases. However, the localization pattern of CD44 in membrane of PV patients appeared to be distorted as compared with normal skin, suggesting a possible role of CD44 in this disease. More experimentation is needed to fully understand the role of CD44 in normal skin barrier function and how it is affected in PV.
|
| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
My former supervisor, the Professor and Chairman of our research institute was scheduled to retire from the organization in March 2017. Due to his impending retirement, there were other urgent things that needed to be completed before his retirement. Therefore, most of the research effort went into completing the other urgent activities to enable my Professor clear up those outstanding tasks before his retirement. For these reason, the research has delayed slightly. The new environment is now well suited to continue with the research and the research will be expected to run smoothly from now on.
|
| Strategy for Future Research Activity |
Specific cDNAs and siRNA have been cloned into vectors for over expression and down regulation of CD44 in human keratinocytes. These vectors will be introduced into human keratinocytes using xfect reagent or lentiviral delivery system and the effects of their over-expression or down-regulation will be examined in normal and malignant keratinocytes. Primary keratinocytes or keratinocyte cell lines will be purchased. RNA will be prepared from these cells carrying specific CD44 cDNA or siRNA under different culture conditions and subjected to Micro-array analysis to identify novel pathways controlled by specific CD44 molecules under various normal and pathological conditions. Various analytical methods such as Western blotting, immunofluorescence, ELISA, RT-PCR, etc will be required to monitor the fate of CD44 proteins and RNA in human keratinocytes under various cell culture conditions. Furthermore, expression of CD44 in various skin diseases will be examined. Finally, whether autoantibodies are produced against CD44 molecules in human skin autoimmune diseases will be investigated.
|
| Causes of Carryover |
The research is still ongoing and there are many reagents and cells that needed to be purchased in order to continue with the research.
|
| Expenditure Plan for Carryover Budget |
The grant will be used to purchase reagents that are needed to perform gene expression analysis including reagents for Western blotting, immunofluorescence ELISA, etc. These include primary and secondary antibodies as well as substrates for detection of signals. Primary keratinocyte cells or keratinocyte cell lines will also be purchased. Media and cell culture related reagents will be purchased. After introducing the cDNA or siRNA into cells, RNA will be subjected to Micro-array analysis. Part of the grant will be used to pay for Micro-array analysis services. A small part of the grant will also be used to travel to present the results of the research at academic meetings.
|
-
[Journal Article] Isolation of All CD44 Transcripts in Human Epidermis and Regulation of Their Expression by Various Agents.2016
Author(s)
Kwesi Teye, Sanae Numata, Norito Ishii, Rafal P. Krol, Atsunari Tsuchisaka, Takahiro Hamada, Hiroshi Koga, Tadashi Karashima, Chika Ohata, Daisuke Tsuruta, Hideyuki Saya, Marek Haftek, Takashi Hashimoto
-
Journal Title
PLoS ONE
Volume: 11(8)
Pages: e0160952
DOI
Peer Reviewed / Open Access / Int'l Joint Research
-
