2015 Joint Usage and Research Report
Title of Research Project
Institution
ApplicantNational Institute of Dental and Craniofacial Research,
Molecular Physiology and Therapeutics Branch

Job title

Senior Investigator and Deputy Branch Chiefand name
Visiting

Name

researcher
Purpose of the Research Project(approx. 250 words)

John

Chiorini

A major problem facing medicine today is defining themechanism associated with the onset and symptoms of disease
and developing treatment for them. With the reported increasein the prevalence and incidence of autoimmune diseases thisgroup of diseases represents a major health concern. Sjögren’s
syndrome is the second most common autoimmune disease andis estimated to affect over 35 million individuals worldwide.
This disease is characterized by a poor quality of life due tosecretory epithelial cell dysfunction with dry mouth and dryeyes, lymphocytic infiltration within the affected salivary glandtissue, development of autoantibodies, and a 30 fold increased
risk of developing lymphoma. Currently, no effective treatment
exists for Sjögren’s syndrome and our understanding of thephysiological mechanism associated with the xerostomia is notclear. Analysis of the transcriptome can provide a global
perspective of the physiologic state of the cell and identify
specific as well as broad changes within the cell. Our lab has
utilized this approach to investigate central questions related to
Sjögren’s syndrome such as the gene expression changes
associated with the loss of gland activity. Through thisanalysis we have recently discovered many cellular changes in
the gland associate with Akt signaling and autophagy.
Correspondingly Dr. Noguchi’ s laboratory has discovered bymeans of yeast two hybrid screening using full-length Akt as abait, a connection with critical proteins in the autophagy
pathway, specifically Phafin2. The collaborative work with Dr.
Noguchi’s laboratory should clarify the role of Akt and theinduction of autophagy in the initiation and progression of
Sjogren’s syndrome.
In this research proposal, we will use recombinant DNA
experiments as well as animal studies.

Development of the Research
Project and Results(approx.. 850 words)

Autophagy is originally characterized as a conserved process
by which cells recycle cytoplasm and defective organelles (i.e.
mitochondria) in diverged organisms from yeast to mammals.
The process is associated with degradation and recycling during
stress situations such as nutrient starvation (Klionsky, 2005;
Levine et al., 2011; Mizushima et al., 2008). Recent study
demonstrated that autophagy plays an important role in vivo
physiology, so that malfunctioning of autophagy underlies
various pathological condition including autoimmune diseases
such as primary Sjögren syndrome (pSS) (Klionsky, 2005;
Levine et al., 2011; Mizushima et al., 2008).
We have previously shown that phafin2 associated Akt cantranslocalize to the lysosome, which is critical for induction ofautophagy(Matsuda-Lennikov et al., 2014). This observationstrongly supports that Akt can play an important role in theinduction of autophagy. In an effort to identify the molecules
underlie the pathogenesis of Sjögren syndrome, we conducted
DNA microarray analysis to identify differentially expressed
genes in Sjögren syndrome patients. We found that LAMP3,(lysosomal associated protein 3, formerly called Dendric CellLAMP) was enriched over 5 fold in pSS.
In our effort to work with Dr. Noguchi’ s laboratory using
lenti-virus expression systems, we overexpressed LAMP3 in
HT1080 cells, and measured the level of autophagic induction
by LC3 western blot. We found that LAMP3 overexpression
clearly induce
autophagy.
Moreover, LAMP3
physically associatedwith Akt in
co-immunoprecipitation
assay in HEK293 cells.
These observation
supported the potential
role of LAMP3 in thepathogenesis of pSS.
Method: HT1080 cells
were transduced byLentiviral
HA-LAMP3transduction(pCSII-CMV-IRES2-Venus). Cells were treated with HBSS orRapamycin to induce autophagy and harvested and subjected toimmunoblot with LC3 antibody.
In our contining international collaboration with Dr. Noguchi,
we will further clarify the functional role of LAMP3-Aktinteraction as an underlying mechanisms of pSS. Given the highfrequency of clinical association of lymphoma in pSS, functional
interaction of the Akt-LAMP3, which is overexpressed in pSSpatients, may represent a critical molecular mechanisms
associated with this complication.

Klionsky, D.J. (2005). The molecular machinery of autophagy:unanswered questions. J Cell Sci 118, 7-18.
Levine, B., Mizushima, N., and Virgin, H.W. (2011). Autophagy inimmunity and inflammation. Nature 469, 323-335.
Matsuda-Lennikov, M., Suizu, F., Hirata, N., Hashimoto, M.,Kimura, K., Nagamine, T., Fujioka, Y., Ohba, Y., Iwanaga, T., andNoguchi, M. (2014). Lysosomal interaction of akt with phafin2: acritical step in the induction of autophagy. PloS one 9, e79795.
Mizushima, N., Levine, B., Cuervo, A.M., and Klionsky, D.J. (2008).
Autophagy fights disease through cellular self-digestion. Nature451, 1069-1075.

Publication【Conference, symposium, workshop etc.】*Enter the names of conferenceor journal and its vol. No. where
the above work was presented.
【Journals】