title: Functional characterization of SAMD9, a protein deficient in normophosphatemic familial tumoral calcinosis.
abstract: Dystrophic cutaneous calcinosis is associated with disorders as common as autoimmune diseases and cancer. To get insight into the pathogenesis of this poorly understood process, we studied the function of SAMD9, a protein of unknown function, recently shown to be deficient in a hereditary form of dystrophic calcification in the skin, known as normophosphatemic familial tumoral calcinosis (NFTC). Consistent with the fact that in NFTC severe inflammatory manifestations always precede cutaneous calcinosis, we found out that SAMD9 is tightly regulated by interferon-γ (IFN-γ). In addition, the SAMD9 promoter was also found to respond strongly to IFN-γ in a luciferase reporter assay. Of interest, we identified a critical 30-bp fragment upstream to the SAMD9 transcription initiation site responsible for driving most of the gene expression. Bioinformatic analysis suggested that SAMD9 function involves interaction with additional protein(s). Using the Ras recruitment system assay and confirmatory immunoprecipitation, we demonstrated that SAMD9 interacts with RGL2. To study the biological importance of this interaction, we assessed the effect of RNA interference-mediated downregulation of this pair of proteins in various cell lines. We found out that downregulation of any of the two protein partners caused increased expression of EGR1, a transcription factor with a known role in the regulation of tissue calcification, inflammation, and cell migration. Supporting the physiological relevance of these data, EGR1 levels were also upregulated in a fibroblast cell line derived from an NFTC patient. In conclusion, our data indicate that SAMD9, an IFN-γ-responsive protein, interacts with RGL2 to diminish the expression of EGR1, a protein of direct relevance to the pathogenesis of ectopic calcification and inflammation.
authors.affiliation: Institute of Pathology, Rambam Health Care Campus, Haifa, Israel.
authors.forename: Dov
authors.initials: D
authors.lastname: Hershkovitz
authors.forename: Yonit
authors.initials: Y
authors.lastname: Gross
authors.forename: Sagi
authors.initials: S
authors.lastname: Nahum
authors.forename: Shiran
authors.initials: S
authors.lastname: Yehezkel
authors.forename: Ofer
authors.initials: O
authors.lastname: Sarig
authors.forename: Jouni
authors.initials: J
authors.lastname: Uitto
authors.forename: Eli
authors.initials: E
authors.lastname: Sprecher
mesh_terms: D002114:Calcinosis / Q000503:physiopathology*; D002478:Cells, Cultured; D051766:Early Growth Response Protein 1 / Q000502:physiology; D005347:Fibroblasts / Q000473:pathology / Q000502:physiology; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D007371:Interferon-gamma / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D012871:Skin Diseases / Q000503:physiopathology*; D020691:rab GTP-Binding Proteins / Q000502:physiology
chemical_list: C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D020662:Guanine Nucleotide Exchange Factors; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; C102739:RGL2 protein, human; C514443:SAMD9 protein, human; D007371:Interferon-gamma; D020691:rab GTP-Binding Proteins