A 4 bp deletion mutation in DLX3 enhances osteoblastic differentiation and bone formation in vitro.

A 4 base-pair deletion mutation in the Distal-less 3 (DLX3) gene is etiologic for Tricho-Dento-Osseous syndrome (TDO). A cardinal feature of TDO is an increased thickness and density of bone. We tested the effects of the DLX3 gene mutation responsible for TDO on the osteoblastic differentiation of preosteoblastic MC3T3E1 cells and multipontent mesenchymal C2C12 cells. Differential expression analysis of C2C12 cells transfected with wild type DLX3 or mutant DLX3 was performed and desmin gene expression, an early myoblastic differentiation marker in mesenchymal cells, was evaluated by RT-PCR, western blot analysis, and desmin promoter transcriptional activity. Transfection of wild type DLX3 into MC3T3E1 and C2C12 cells increased alkaline phosphatase-2 activity, mineral deposition, and promoter activities of the osteocalcin and type 1 collagen genes compared to empty vector transfected cells. Transfection of mutant DLX3 into these cells further enhanced alkaline phosphatase activity, mineral deposition, and osteocalcin promoter activities, but did not further enhance type 1 collagen promoter activity. Transfection of mutant DLX3 into C2C12 cells markedly down regulated desmin gene expression, and protein expression of desmin and MyoD, while increasing protein expression of osterix and Runx2. These results demonstrate that the DLX3 deletion mutation associated with TDO enhances mesenchymal cell differentiation to an osteoblastic lineage rather than a myoblastic lineage by changing the fate of mesenchymal cells. This DLX3 mutation also accelerates the differentiation of osteoprogenitor cells to osteoblasts at later stages of osteogenesis.

Changes in salivary proteome following allogeneic hematopoietic stem cell transplantation.

OBJECTIVE: Allogeneic hematopoietic stem cell transplantation (allo-HCT) is frequently complicated by severe infections and graft-vs-host disease (GVHD). Saliva contains many components of adaptive and innate immune response crucial for local host defenses. Changes in salivary constituents could reflect systemic processes such as immune reconstitution and development of GVHD that occur posttransplant. This study was an initial evaluation of salivary protein changes that occur after allo-HCT. PATIENTS AND METHODS: Serially collected saliva samples from 41 patients undergoing allo-HCT were evaluated. Changes in salivary proteome were initially examined by SELDI-TOF mass spectrometry. Individual protein changes were identified by 2-dimensional differential in-gel electrophoresis (2D-DIGE) with subsequent MS/MS sequencing and ELISA. RESULTS: Significant increases and decreases in multiple salivary proteins that lasted at least 2 months posttransplant were detected by SELDI-TOF mass spectrometry. Lactoferrin and secretory leukocyte protease inhibitor demonstrated elevations 1 month post-HCT that persisted at least 6 months. Secretory IgA (sIgA) levels were decreased 1 month posttransplant, with recovery at approximately 6 months. Levels of salivary beta(2)-microglobulin were elevated at 6 months and correlated with sIgA levels. CONCLUSION: Allo-HCT is associated with long-term changes in several salivary proteins important for innate immune responses. These results support further studies on the association of salivary proteins with posttransplant complications including infections and GVHD.

Human periodontal fibroblast response to enamel matrix derivative, amelogenin, and platelet-derived growth factor-BB.

BACKGROUND: The ideal goal of clinical therapy in periodontal defects is regeneration of all lost structures. For regeneration to occur, cell proliferation, migration, and extracellular matrix synthesis are prerequisites. Attempts at regeneration of periodontal defects by guided tissue regeneration using bone grafts and membranes have not always yielded predictable results. Recently, attempts at engineering the defects using various materials have shown promising results. Two such approaches have been used to regenerate periodontal defects, one using extracellular matrix such as enamel matrix proteins and the other using growth factors. However, to our knowledge, no study has looked at combining these two approaches to achieve potentially even greater regeneration. METHODS: Primary human periodontal ligament (PDL) fibroblasts were explanted, and alkaline phosphatase (ALK PHOS) activity was determined. Phenotypically different cell lines were incubated for 1, 3, 6, and 10 days in 0.2% fetal bovine serum (FBS) media containing different concentrations of either enamel matrix derivative (EMD), amelogenin, platelet-derived growth factor-BB (PDGF-BB), EMD+PDGF-BB, or amelogenin+PDGF-BB. A culture of 0.2% FBS alone served as a negative control, and a culture of 10% FBS served as a positive control. Cell proliferation was measured using a Coulter counter to determine the cell number. The effects on a wound-fill model were evaluated by scraping a 3-mm wide cell-free zone in PDL monolayers across the diameter of the tissue-culture plate and determining PDL cell migration into the cell-free zone using computer assisted histomorphometry. RESULTS: Compared to the control, only EMD+PDGF-BB significantly increased PDL cell proliferation in an ALK PHOS (-) cell line (P<0.001), and EMD alone, EMD+PDGF-BB, and amelogenin+PDGF-BB significantly increased PDL cell proliferation in an ALK PHOS (+) cell line (P<0.001) with EMD+PDGF-BB showing a trend for greater proliferation than either PDGF or EMD alone. Individually, EMD and amelogenin had no significant effect on PDL cell proliferation. In the wound-fill experiment, all factors and their combinations except amelogenin significantly enhanced cell migration compared to the control (P<0.05) at the wound edge. In addition, EMD+PDGF-BB had additive effects on the ALK PHOS (-) cell line at the wound edge. At the center of the wound, neither EMD nor amelogenin had a significant wound-fill effect. However, the combination of EMD+PDGF-BB additively increased wound fill for both ALK PHOS (+) and ALK PHOS (-) cells. CONCLUSIONS: The combination of EMD and PDGF-BB produces greater proliferative and wound-fill effects on PDL cells than each by themselves. If these combined effects can be translated clinically, one may see greater regeneration in periodontal defects with this combination. However, amelogenin does not have significant effects on PDL cell proliferation or migration by itself. This may suggest that either another enamel matrix component in EMD may be responsible for some of its clinical effects, or that amelogenin alone may not trigger the regenerative potential of periodontal tissues and that it requires a combined interaction with other enamel matrix components of EMD to direct the regenerative process.

Clinical, genetic, and biochemical findings in two siblings with Papillon-Lefèvre Syndrome.

BACKGROUND: Papillon-Lefèvre Syndrome (PLS) is an autosomal recessive disease characterized by palmoplantar hyperkeratosis and severe periodontitis affecting both primary and secondary dentitions. Cathepsin C (CTSC) gene mutations are etiologic for PLS. The resultant loss of CTSC function is responsible for the severe periodontal destruction seen clinically. METHODS: A 4-year-old female (case 1) and her 10-year-old sister (case 2) presented with palmoplantar skin lesions, tooth mobility, and advanced periodontitis. Based on clinical findings, the cases were diagnosed with PLS. Mutational screening of the CTSC gene was conducted for the cases, and their clinically unaffected parents and brother. Biochemical analysis was performed for CTSC, cathepsin G (CTSG), and elastase activity in neutrophils for all members of the nuclear family. The initial treatment included oral hygiene instruction, scaling and root planing, and systemic amoxicillin-metronidazole therapy. RESULTS: CTSC mutational screening identified a c.415G>A transition mutation. In the homozygous state, this mutation was associated with an almost complete loss of activity of CTSC, CTSG, and elastase. Although monthly visits, including scaling, polishing, and 0.2% chlorhexidine digluconate irrigation were performed to stabilize the periodontal condition, case 1 lost all her primary teeth. In case 2, some of the permanent teeth could be maintained. CONCLUSIONS: This report describes two siblings with a cathepsin C gene mutation that is associated with the inactivity of cathepsin C and several neutrophil serine proteases. The failure of patients to respond to periodontal treatment is discussed in the context of these biological findings.

Mutant tamm-horsfall glycoprotein accumulation in endoplasmic reticulum induces apoptosis reversed by colchicine and sodium 4-phenylbutyrate.

As a consequence of uromodulin gene mutations, individuals develop precocious hyperuricemia, gout, and progressive renal failure. In vitro studies suggest that pathologic accumulation of uromodulin/Tamm-Horsfall glycoprotein (THP) occurs in the endoplasmic reticulum (ER), but the pathophysiology of renal damage is unclear. It was hypothesized that programmed cell death triggered by accumulation of misfolded THP in the ER causes progressive renal disease. Stably transfected human embryonic kidney 293 cells and immortalized thick ascending limb of Henle's loop cells with wild-type and mutated uromodulin cDNA were evaluated to test this hypothesis. Immunocytochemistry, ELISA, and deglycosylation studies indicated that accumulation of mutant THP occurred in the ER. FACS analyses showed a significant increase in early apoptosis signal in human embryonic kidney 293 and thick ascending limb of Henle's loop cells that were transfected with mutant uromodulin constructs. Colchicine and sodium 4-phenylbutyrate treatment increased secretion of THP from the ER to the cell membrane and into the culture media and significantly improved cell viability. These findings indicate that intracellular accumulation of THP facilitates apoptosis and that this may provide the pathologic mechanism responsible for the progressive renal damage associated with uromodulin gene mutations. Colchicine and sodium 4-phenylbutyrate reverse these processes and could potentially be beneficial in ameliorating the progressive renal damage in uromodulin-associated kidney diseases.

Proteolysis of macrophage inflammatory protein-1alpha isoforms LD78beta and LD78alpha by neutrophil-derived serine proteases.

Macrophage inflammatory protein-1alpha (MIP-1alpha) is a chemokine that leads to leukocyte recruitment and activation at sites of infection. Controlling chemokine activity at sites of infection is important, since excess accumulation of leukocytes may contribute to localized tissue damage. Neutrophil-derived serine proteases modulate the bioactivity of chemokine and cytokine networks through proteolytic cleavage. Because MIP-1alpha is temporally expressed with neutrophils at sites of infection, we examined proteolysis of MIP-1alpha in vitro by the neutrophil-derived serine proteases: cathepsin G, elastase, and proteinase 3. Recombinant human MIP-1alpha isoforms LD78beta and LD78alpha were expressed and purified, and the protease cleavage sites were analyzed by mass spectrometry and peptide sequencing. Chemotactic activities of parent and cleavage molecules were also compared. Both LD78beta and LD78alpha were cleaved by neutrophil lysates at Thr16-Ser17, Phe24-Ile25, Tyr28-Phe29, and Thr31-Ser32. This degradation was inhibited by serine protease inhibitors phenylmethylsulfonyl fluoride and 4-(2-aminoethyl)-benzenesulfonyl fluoride. Incubation of the substrates with individual proteases revealed that cathepsin G preferentially cleaved at Phe24-Ile25 and Tyr28-Phe29, whereas elastase and proteinase 3 cleaved at Thr16-Ser17 and Thr31-Ser32. Proteolysis of LD78beta resulted in loss of chemotactic activity. The role of these proteases in LD78beta and LD78alpha degradation was confirmed by incubation with neutrophil lysates from Papillon-Lefevre syndrome patients, demonstrating that the cell lysates containing inactivated serine proteases could not degrade LD78beta and LD78alpha. These findings suggest that severe periodontal tissue destruction in Papillon-Lefevre syndrome may be related to excess accumulation of LD78beta and LD78alpha and dysregulation of the microbial-induced inflammatory response in the periodontium.