MicroRNAs (miRNA) are short non-coding RNA molecules that regulate a variety of biological processes. The role of miRNAs in BMP2-mediated biological processes is of considerable interest. A comparative miRNA array led to the isolation of several BMP2-responsive miRNAs. Among them, miR-199a* is of particular interest, because it was reported to be specifically expressed in the skeletal system. Here we demonstrate that miR199a* is an early responsive target of BMP2: its level was dramatically reduced at 5 h, quickly increased at 24 h and remained higher thereafter in the course of BMP2-triggered chondrogenesis of a micromass culture of pluripotent C3H10T1/2 stem cells. miR-199a* significantly inhibited early chondrogenesis, as revealed by the reduced expression of early marker genes for chondrogenesis such as cartilage oligomeric matrix protein (COMP), type II collagen, and Sox9, whereas anti-miR-199a* increased the expression of these chondrogenic marker genes. A computer-based prediction algorithm led to the identification of Smad1, a well established downstream molecule of BMP-2 signaling, as a putative target of miR-199a*. The pattern of Smad1 mRNA expression exhibited the mirror opposite of miR199a* expression following BMP-2 induction. Furthermore, miR-199a* demonstrated remarkable inhibition of both endogenous Smad1 as well as a reporter construct bearing the 3-untranslated region of Smad1 mRNA. In addition, mutation of miR-199a* binding sites in the 3-untranslated region of Smad1 mRNA abolished miR-199a*-mediated repression of reporter gene activity. Mechanism studies revealed that miR-199a* inhibits Smad1/Smad4-mediated transactivation of target genes, and that overexpression of Smad1 completely corrects miR-199a*-mediated repression of early chondrogenesis. Taken together, miR-199a* is the first BMP2 responsive microRNA found to adversely regulate early chondrocyte differentiation via direct targeting of the Smad1 transcription factor. MicroRNAs (miRNAs)3 are a class of short (ϳ20 -24 nucleotide) non-coding single-stranded RNA molecules that are important regulators of cellular gene expression. First discovered in 1993, they are thought to regulate the expression of approximately one-third of all mammalian genes (1). Functioning at the post-transcriptional level, miRNAs inhibit mRNA expression by binding to the 3Ј-untranslated region (3Ј-UTR) of mRNA before directing the repression of translation and/or mRNA degradation. They have been implicated as important regulators of a variety of biological processes including cell proliferation, differentiation, development, and tumorigenesis (2-9).Mature single-stranded miRNA is generated from a long primary genomic transcript (pri-miRNA), which is processed in the nucleus by the enzymes Drosha and DGCR8, resulting in the excision of a stem loop structure. The resulting 60 -80-nucleotide precursor miRNA (pre-miRNA) is exported into the cytoplasm by Exportin 5. In the cytoplasm, the RNase III enzyme Dicer processes the precursor miRNA to generate a short RNA duple...

Depletional induction therapies are routinely used to prevent acute rejection and improve transplant outcome. The effects of depleting agents on T-cell subsets and subsequent T-cell reconstitution are incompletely defined. We used flow cytometry to examine the effects of rabbit antithymocyte globulin (rATG) on the peripheral T-cell repertoire of pediatric and adult renal transplant recipients. We found that while rATG effectively depleted CD45RA+CD27+ naïıve and CD45RO+CD27+ central memory CD4+ T cells, it had little effect on CD45RO+CD27− CD4+ effector memory or CD45RA+CD31−, CD45RO+CD27+ and CD45RO+CD27− CD8+ T cell subsets. When we performed a kinetic analysis of CD31+ recent thymic emigrants and CD45RA+/RO+ T cells, we found evidence for both thymopoiesis and homeostatic proliferation contributing to immune reconstitution.We additionally examined the impact of rATG on peripheral CD4+Foxp3+ T cells.We found that in adults, administration of rATG-induced peripheral expansion and new thymic emigration of T cells with a Treg phenotype, while CD4+Foxp3+ T cells of thymic origin predominated in children, providing the first evidence that rATG induces Treg in vivo. Collectively our data indicate that rATG alters the balance of regulatory to memory effector T cells posttransplant, providing an explanation for how it positively impacts transplant outcome.

Loss of articular cartilage because of extracellular matrix breakdown is the hallmark of arthritis. Degradative fragments of cartilage oligomeric matrix protein (COMP), a prominent noncollagenous matrix component in articular cartilage, have been observed in the cartilage, synovial fluid, and serum of arthritis patients. The molecular mechanism of COMP degradation and the enzyme(s) responsible for it, however, remain largely unknown. ADAMTS-12 (a disintegrin and metalloprotease with thrombospondin motifs) was shown to associate with COMP both in vitro and in vivo. ADAMTS-12 selectively binds to only the epidermal growth factorlike repeat domain of COMP of the four functional domains tested. The four C-terminal TSP-1-like repeats of ADAMTS-12 are shown to be necessary and sufficient for its interaction with COMP. Recombinant ADAMTS-12 is capable of digesting COMP in vitro. The COMP-degrading activity of ADAMTS-12 requires the presence of Zn 2؉ and appropriate pH (7.5-9.5), and the level of ADAMTS-12 in the cartilage and synovium of patients with both osteoarthritis and rheumatoid arthritis is significantly higher than in normal cartilage and synovium. Together, these findings indicate that ADAMTS-12 is a new COMP-interacting and -degrading enzyme and thus may play an important role in the COMP degradation in the initiation and progression of arthritis.More than 15% of the world population older than 18 years are affected by arthritic disorders, including osteoarthritis (OA) 3 and rheumatoid arthritis (RA) (1). Accumulating evidence suggests that proteases perform an important function in the breakdown of the extracellular matrix in OA and RA (2). Cartilage oligomeric matrix protein (COMP), a prominent noncollagenous component of cartilage, accounts for ϳ1% of the wet weight of articular tissue (3, 4). COMP is a 524-kDa pentameric, disulfide-bonded, multidomain glycoprotein composed of approximately equal subunits (ϳ110 kDa each) (5, 6). Several studies suggest that monitoring of COMP levels (in both joint fluid and serum) can be used to assess the presence and progression of arthritis (7-11). Synovial fluid COMP levels were found to be higher in individuals with knee pain or injury (12), anterior cruciate ligament or meniscal injury (9, 12), OA (8, 12), and RA (7, 13) than in healthy individuals.Fragments of COMP have been detected in the cartilage, synovial fluid, and serum of patients with post-traumatic and primary OA and RA (7,8,13). The molecular mechanism of COMP degradation and the enzyme (s) responsible for it, however, remain largely unknown. Theoretically, inhibition of degradative enzymes can slow down or block the initiation and progression of arthritic diseases. The isolation of cartilage degradative enzymes is therefore of great interest from both a pathophysiological and a therapeutic standpoint. The ADAMTS family (ADAMTS: (a disintegrin and metalloprotease with thrombospondin motifs) consists of secreted zinc metalloproteinases with a precisely ordered modular organization that includes at least o...

Objective-As we previously reported, ADAMTS-7 and ADAMTS-12, two members of ADAMTS (adisintegrin and metalloprotease with thrombospondin motifs) family, degrade COMP in vitro and are significantly induced in the cartilage and synovium of arthritic patients. The purpose of this study was to determine 1) whether cleavage activity of ADAMTS-7 and-12 of COMP are associated with COMP degradation in osteoarthritis; 2) whether a2M is a novel substrate for ADAMTS-7 and-12; and 3) whether a2M inhibits ADAMTS-7 or-12 cleavage of COMP. Methods-An in vitro digestion assay was used to examine the degradation of COMP by ADAMTS-7 and ADAMTS-12 in the cartilage of OA patients; in cartilage explants incubated with TNF-α or IL-1β with or without blocking antibodies; and in human chondrocytes treated with specific siRNA to knock down ADAMTS-7 or/and-12. Digestion of alpha-2-macroglobulin (a2M) by ADAMTS-7 and-12 in vitro and the inhibition of ADAMTS-7 or-12-mediated digestion of COMP by α2M were also analyzed. Results-The molecular mass of the COMP fragments produced by either ADAMTS-7 or ADAMTS-12 were similar to those observed in OA patients. Specific blocking antibodies against ADAMTS-7 and ADAMTS-12 dramatically inhibited TNF-α-or IL-1β-induced COMP degradation in the cultured cartilage explants. The suppression of ADAMTS-7 or ADAMTS-12 expression by siRNA silencing in the human chondrocytes also prevented TNF-α-or IL-1β-induced COMP degradation. Both ADAMTS-7 and ADAMTS-12 were able to cleave α 2 M, giving rise to 180 and 105 kDa cleavage products, respectively. Furthermore, α 2 M inhibited both ADAMTS-7-and ADAMTS-12-mediated COMP degradation in a concentration (or dose)-dependent manner. Conclusion-Our observations demonstrate the importance of COMP degradation by ADAMTS-7 and ADAMTS-12 in vivo. Furthermore, α 2 M is a novel substrate for ADAMTS-7 and ADAMTS-12. More significantly, α 2 M represents the first endogenous inhibitor of ADAMTS-7 and ADAMTS-12.

Myeloid‐derived suppressor cells (MDSC) are negative regulators of the immune response and are in part responsible for the inhibition of the T cell–mediated immune responses. While MDSC have been demonstrated to participate in the induction of prolonged allograft survival in animal models of transplantation, little is known about their immune regulatory function in human transplant recipients. Here, we report that two subsets of human MDSC expressing CD11b+, CD33+ and HLA‐DR− develop in renal patients posttransplantation. We found that CD14+ expressing monocytic MDSC isolated from transplant recipients were highly efficient in suppressing the proliferation of CD4+ T cells in mixed leukocyte reactions. In addition, we observed that CD11b+CD33+HLA‐DR− MDSC are capable of expanding Treg in vitro, and their accumulation overtime after transplantation linearly correlated with an increase in Treg in vivo. This is the first study to link the presence of MDSC with the emergence of Treg in vivo in transplant recipients, and to define the subpopulation of MDSC derived from transplant recipients responsible for generation of Treg. Further studies are necessary to determine the alloimmune regulatory function of MDSC in human transplant recipients.

Summary Background Chronic injury in kidney transplants remains a major cause of allograft loss. The aim of this study was to identify a gene set capable of predicting renal allografts at risk of progressive injury due to fibrosis. Methods This Genomics of Chronic Allograft Rejection (GoCAR) study is a prospective, multicentre study. We prospectively collected biopsies from renal allograft recipients (n=204) with stable renal function 3 months after transplantation. We used microarray analysis to investigate gene expression in 159 of these tissue samples. We aimed to identify genes that correlated with the Chronic Allograft Damage Index (CADI) score at 12 months, but not fibrosis at the time of the biopsy. We applied a penalised regression model in combination with permutation-based approach to derive an optimal gene set to predict allograft fibrosis. The GoCAR study is registered with ClinicalTrials.gov, number NCT00611702. Findings We identified a set of 13 genes that was independently predictive for the development of fibrosis at 1 year (ie, CADI-12 ≥2). The gene set had high predictive capacity (area under the curve [AUC] 0·967), which was superior to that of baseline clinical variables (AUC 0·706) and clinical and pathological variables (AUC 0·806). Furthermore routine pathological variables were unable to identify which histologically normal allografts would progress to fibrosis (AUC 0·754), whereas the predictive gene set accurately discriminated between transplants at high and low risk of progression (AUC 0·916). The 13 genes also accurately predicted early allograft loss (AUC 0·842 at 2 years and 0·844 at 3 years). We validated the predictive value of this gene set in an independent cohort from the GoCAR study (n=45, AUC 0·866) and two independent, publically available expression datasets (n=282, AUC 0·831 and n=24, AUC 0·972). Interpretation Our results suggest that this set of 13 genes could be used to identify kidney transplant recipients at risk of allograft loss before the development of irreversible damage, thus allowing therapy to be modified to prevent progression to fibrosis. Funding National Institutes of Health.

ADAMTS-7, a metalloproteinase that belongs to ADAMTS family, is important for the degradation of cartilage extracellular matrix proteins in arthritis. Herein we report that ADAMTS-7 is upregulated during chondrocyte differentiation and demonstrates the temporal and spatial expression pattern during skeletal development. ADAMTS-7 potently inhibits chondrocyte differentiation and endochondral bone formation, and this inhibition depends on its proteolytic activity. The cysteine-rich domain of ADAMTS-7 is required for its interaction with the extracellular matrix, and the C-terminal four-thrombospondin motifs are necessary for its full proteolytic activity and inhibition of chondrocyte differentiation. ADAMTS-7 is an important target of canonical PTHrP signaling, since (i) PTHrP induces ADAMTS-7, (ii) ADAMTS-7 is downregulated in PTHrP null mutant (PTHrP؊/؊) growth plate chondrocytes, and (iii) blockage of ADAMTS-7 almost abolishes PTHrP-mediated inhibition of chondrocyte hypertrophy and endochondral bone growth. ADAMTS-7 associates with granulin-epithelin precursor (GEP), an autocrine growth factor that has been implicated in tissue regeneration, tumorigenesis, and inflammation. In addition, ADAMTS-7 acts as a new GEP convertase and neutralizes GEP-stimulated endochondral bone formation.Collectively, these findings demonstrate that ADAMTS-7, a direct target of PTHrP signaling, negatively regulates endochondral bone formation by associating with and inactivating GEP chondrogenic growth factor.The ADAMTS family consists of secreted zinc metalloproteinases with a precisely ordered modular organization that includes at least one thrombospondin type I repeat (51, 53). Important functions have been established for several members of the ADAMTS family. ADAMTS-1, ADAMTS-4, ADAMTS-5, ADAMTS-8, ADAMTS-9, ADAMTS-16, and ADAMTS-18 degrade the cartilage aggrecan (1,19,36,61,84,88), and ADAMTS-5 plays a primary role in aggrecan loss in arthritis (36, 84). ADAMTS-2, ADAMTS-3, and ADAMTS-14 are procollagen N-propeptidases (18, 30). ADAMTS-2 mutations cause dermatosparaxis, an inherited disorder characterized by severe skin fragility (17). ADAMTS-13 is a von Willebrand factor-cleaving protease, and its mutations lead to heritable life-threatening thrombocytopenic purpura (65). ADAMTS-12 and ADAMTS-7 share the same domain organization and structure and form a subgroup within the ADAMTS family (13, 83). These two enzymes have been found to associate with alpha-2-macroglobulin (13,70,83), and ADAMTS-12 also degrades aggrecan (68). Studies from our group demonstrated that ADAMTS-7 and ADAMTS-12 directly associate with and degrade COMP, a prominent noncollagenous component of cartilage (66,67). COMP is a 524-kDa, pentameric, disulfide-bonded, multidomain glycoprotein composed of approximately equal subunits (ϳ110 kDa each) (43,75). Although the function of COMP is not completely understood, it appears to mediate chondrocyte attachment by an integrin receptor (15,29), and accumulating evidence suggests that COMP may function to stabilize...

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