Failure to Process Dentin Matrix Protein 1 (DMP1) into Fragments Leads to Its Loss of Function in Osteogenesis

Dentin matrix protein 1 (DMP1), an acidic protein important to the formation of bone and dentin, primarily exists as the processed NH₂-terminal and COOH-terminal fragments in the extracellular matrix of the two tissues. Previous in vitro studies showed that the substitution of residue Asp²¹³ by Ala²¹³ (D213A) at a cleavage site blocked the processing of mouse DMP1 in cells. In this study, we generated transgenic mice expressing mutant D213A-DMP1 (WT/D213A-Tg mice) to test the hypothesis that the proteolytic processing of DMP1 is an activation step essential to osteogenesis. By crossbreeding WT/D213A-Tg mice with Dmp1 knock-out (Dmp1-KO) mice, we obtained mice expressing D213A-DMP1 in a Dmp1-KO background; these mice will be referred to as “Dmp1-KO/D213A-Tg” mice. Biochemical, radiological, and morphological approaches were used to characterize the skeletal phenotypes of Dmp1-KO/D213A-Tg mice compared with wild-type mice, Dmp1-KO mice, and Dmp1-KO mice expressing the normal Dmp1 transgene. Protein chemistry analyses showed that DMP1 was barely cleaved in the bone of the Dmp1-KO/D213A-Tg mice, indicating that D213A substitution effectively blocked the proteolytic processing of DMP1 in vivo. While the expression of the normal Dmp1 transgene completely rescued the phenotypic skeletal changes of the Dmp1-KO mice, the expression of the mutant D213A-Dmp1 transgene failed to do so. These results indicate that the full-length form of DMP1 is an inactive precursor and its proteolytic processing is an activation step essential to the biological functions of this protein in osteogenesis.     

Crystal Structure of the Ubiquitin-like Domain-CUT Repeat-like Tandem of Special AT-rich Sequence Binding Protein 1 (SATB1) Reveals a Coordinating DNA-binding Mechanism

SATB1 is essential for T-cell development and growth and metastasis of multitype tumors and acts as a global chromatin organizer and gene expression regulator. The DNA binding ability of SATB1 plays vital roles in its various biological functions. We report the crystal structure of the N-terminal module of SATB1. Interestingly, this module contains a ubiquitin-like domain (ULD) and a CUT repeat-like (CUTL) domain (ULD-CUTL tandem). Detailed biochemical experiments indicate that the N terminus of SATB1 (residues 1–248, SATB1^(1–248)), including the extreme 70 N-terminal amino acids, and the ULD-CUTL tandem bind specifically to DNA targets. Our results show that the DNA binding ability of full-length SATB1 requires the contribution of the CUTL domain, as well as the CUT1-CUT2 tandem domain and the homeodomain. These findings may reveal a multiple-domain-coordinated mechanism whereby SATB1 recognizes DNA targets.     

Phospholipid Scramblase 1 Is Secreted by a Lipid Raft-dependent Pathway and Interacts with the Extracellular Matrix Protein 1 in the Dermal Epidermal Junction Zone of Human Skin

We examined the interaction of ECM1 (extracellular matrix protein 1) using yeast two-hybrid screening and identified the type II transmembrane protein, PLSCR1 (phospholipid scramblase 1), as a binding partner. This interaction was then confirmed by in vitro and in vivo co-immunoprecipitation experiments, and additional pull-down experiments with GST-tagged ECM1a fragments localized this interaction to occur within the tandem repeat region of ECM1a. Furthermore, immunohistochemical staining revealed a partial overlap of ECM1 and PLSCR1 in human skin at the basal epidermal cell layer. Moreover, in human skin equivalents, both proteins are expressed at the basal membrane in a dermal fibroblast-dependent manner. Next, immunogold electron microscopy of ultrathin human skin sections showed that ECM1 and PLSCR1 co-localize in the extracellular matrix, and using antibodies against ECM1 or PLSCR1 cross-linked to magnetic immunobeads, we were able to demonstrate PLSCR1-ECM1 interaction in human skin extracts. Furthermore, whereas ECM1 is secreted by the endoplasmic/Golgi-dependent pathway, PLSCR1 release from HaCaT keratinocytes occurs via a lipid raft-dependent mechanism, and is deposited in the extracellular matrix. In summary, we here demonstrate that PLSCR1 interacts with the tandem repeat region of ECM1a in the dermal epidermal junction zone of human skin and provide for the first time experimental evidence that PLSCR1 is secreted by an unconventional secretion pathway. These data suggest that PLSCR1 is a multifunctional protein that can function both inside and outside of the cell and together with ECM1 may play a regulatory role in human skin.     

Lipopolysaccharide Decreases Single Immunoglobulin Interleukin-1 Receptor-related Molecule (SIGIRR) Expression by Suppressing Specificity Protein 1 (Sp1) via the Toll-like Receptor 4 (TLR4)-p38 Pathway in Monocytes and Neutrophils

Single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR) is one of the immunoglobulin-like membrane proteins that is crucial for negative regulation of toll-like receptor 4 (TLR4) and interleukin-1 receptor. Despite the importance of understanding its expression and function, knowledge is limited on the regulatory mechanism in the epithelial tissues, such as the liver, lung, and gut, where its predominant expression is originally described. Here, we found expression of SIGIRR in non-epithelial innate immune cells, including primary peripheral blood monocytes, polymorphonuclear neutrophils, monocytic RAW264 cells, and neutrophilic-differentiated HL-60 cells. Consistent with previous findings in epithelial tissues, SIGIRR gene and protein expression were also down-regulated by LPS treatment in a time-dependent manner in primary blood monocytes and polymorphonuclear neutrophils. A reduction was also observed in RAW264 and differentiated HL-60 cells. Notably, exogenous introduction of the dominant negative form of TLR4 and siRNA of p38 resulted in inhibition of LPS-induced SIGIRR down-regulation, whereas treatment with p38 activator anisomycin showed a dose-dependent decrease in SIGIRR expression, suggesting TLR4-p38 signal as a critical pathway for LPS-induced SIGIRR down-regulation. Finally, reporter gene and chromatin immunoprecipitation assays demonstrated that Sp1 is a key factor that directly binds to the proximal promoter of SIGIRR gene and consequently regulates basal SIGIRR expression, which is negatively regulated by the LPS-dependent TLR4-p38 pathway. In summary, the data precisely demonstrate how LPS down-regulates SIGIRR expression and provide a role of LPS signal that counteracts Sp1-dependent basal promoter activation of SIGIRR gene via TLR4-p38 pathway in non-epithelial innate immune cells.     

Roles for the Mitogen-activated Protein Kinase (MAPK) Phosphatase,DUSP1, in Feedback Control of Inflammatory Gene Expression and Repression by Dexamethasone

Glucocorticoids act on the glucocorticoid receptor (NR3C1) to repress inflammatory gene expression. This is central to their anti-inflammatory effectiveness and rational improvements in therapeutic index depend on understanding the mechanism. Human pulmonary epithelial A549 cells were used to study the role of the mitogen-activated protein kinase (MAPK) phosphatase, dual-specificity phosphatase 1 (DUSP1), in the dexamethasone repression of 11 inflammatory genes induced, in a MAPK-dependent manner, by interleukin-1β (IL1B). Adenoviral over-expression of DUSP1 inactivated MAPK pathways and reduced expression of all 11 inflammatory genes. IL1B rapidly induced DUSP1 expression and RNA silencing revealed a transient role in feedback inhibition of MAPKs and inflammatory gene expression. With dexamethasone, which induced DUSP1 expression, plus IL1B (co-treatment), DUSP1 expression was further enhanced. At 1 h, this was responsible for the dexamethasone inhibition of IL1B-induced MAPK activation and CXCL1 and CXCL2 mRNA expression, with a similar trend for CSF2. Whereas, CCL20 mRNA was not repressed by dexamethasone at 1 h, repression of CCL2, CXCL3, IL6, and IL8 was unaffected, and PTGS2 repression was partially affected by DUSP1 knockdown. At later times, dexamethasone repression of MAPKs was unaffected by DUSP1 silencing. Likewise, 6 h post-IL1B, dexamethasone repression of all 11 mRNAs was essentially unaffected by DUSP1 knockdown. Qualitatively similar data were obtained for CSF2, CXCL1, IL6, and IL8 release. Thus, despite general roles in feedback inhibition, DUSP1 plays a transient, often partial, role in the dexamethasone-dependent repression of certain inflammatory genes. Therefore this also illustrates key roles for DUSP1-independent effectors in mediating glucocorticoid-dependent repression.