Ap Expression Research Articles

TGFβ-mediated inhibition of hypodermal adipocyte progenitor differentiation promotes wound-induced skin fibrosis.

Aberrant activation of dermal fibroblasts during wound healing often leads to debilitating fibrotic changes in the skin, such as scleroderma and keloids. However, the underlying cellular and molecular mechanisms remain elusive. Here, we established a wound-induced skin fibrosis (WISF) mouse model in mature adult mice, characterised by excessive deposition of collagen bundles, loss of dermal adipocytes, and enrichment of DPP4+Ly6A+THY1+ hypodermal interstitial adipocyte progenitors (HI-APs) and pericytes, resembling human fibrotic skin diseases. This WISF model exhibited an age-dependent gain of fibrotic characteristics, contrasting with the wound-induced hair neogenesis observed in younger mice. Through comprehensive analyses of the WISF, we delineated a trajectory of fibroblast differentiation that originates from HI-APs. These progenitors highly expressed several extracellular matrix (ECM) genes and exhibited a TGFβ pathway signature. TGFβ was identified as the key signal to inhibit the adipogenic potential and maintain the fibrogenic potential of dermal APs. Additionally, administering a TGFβ receptor inhibitor to wound scar reduced the abundance of ECM-producing APs. Finally, analysis of human scleroderma skin tissues revealed a negative correlation between the expression of AP-, ECM-, and TGFβ pathway-related genes and PPARG. Overall, this study establishes a wound-induced skin fibrosis mouse model and demonstrates that TGFβ-mediated blockage of HI-AP differentiation is crucial for driving fibrotic pathology. Targeting HI-APs and adipogenesis may provide novel avenues for developing disease-modifying therapies for fibrotic skin diseases.

Comparative Effects of Gymnema sylvestre and Berberine on Adipokines, Body Composition, and Metabolic Parameters in Obese Patients: A Randomized Study.

We performed a comparative study in 50 adult Mexican patients with obesity treated with GS or BBR for 3 months. The baseline and final biochemical parameters, body composition, blood pressure, gene expression of Res, Ome, Vis, and Ap, and safety parameters were evaluated. BBR significantly decreased (p < 0.05) body weight, blood pressure and Vis and Ap gene expression and increased Ome, while GS decreased fasting glucose and Res gene expression (p < 0.05). A comparative analysis of the final measurements revealed a lower gene expression of Ap and Vis (p < 0.05) in patients treated with BBR than in those treated with GS. The most frequent adverse effects in both groups were gastrointestinal symptoms, which attenuated during the first month of treatment. In patients with obesity, BBR has a better effect on body composition, blood pressure, and the gene expression of adipokines related to metabolic risk, while GS has a better effect on fasting glucose and adipokines related to insulin resistance, with minimal side effects.

Impact of Drosophila LIM homeodomain protein Apterous on the morphology of the adult mushroom body

A LIM homeodomain transcription factor Apterous (Ap) regulates embryonic and larval neurodevelopment in Drosophila. Although Ap is still expressed in the adult brain, it remains elusive whether Ap is involved in neurodevelopmental events in the adult brain because flies homozygous for ap mutations are usually lethal before they reach the adult stage. In this study, using adult escapers of ap knockout (KO) homozygotes, we examined whether the complete lack of ap expression affects the morphology of the mushroom body (MB) neurons and Pigment-dispersing factor (Pdf)-positive clock neurons in the adult brain. Although ap KO escapers showed severe structural defects of MB neurons, no clear morphological defects were found in Pdf-positive clock neurons. These results suggest that Ap in the adult brain is essential for the neurodevelopment of specific ap-positive neurons, but it is not necessarily involved in the development of all ap-positive neurons.

The transcription factor optomotor-blind restricts apterous expression through TrxG and PcG genes

The establishment of body pattern is a fundamental process in developmental biology. In Drosophila, the wing disc is subdivided into dorsal (D) and ventral (V) compartments by the D/V boundary. The dorsal fate is adopted by expressing the selector gene apterous (ap). ap expression is regulated by three combinational cis-regulatory modules which are activated by EGFR pathway, Ap-Vg auto-regulatory and epigenetic mechanisms. Here, we found that the Tbx family transcription factor Optomotor-blind (Omb) restricted ap expression in the ventral compartment. Loss of omb induced autonomous initiation of ap expression in the middle third instar larvae in the ventral compartment. Oppositely, over-activation of omb inhibited ap in the medial pouch. All three enhancers apE, apDV and apP were upregulated in omb null mutants, indicating a combinational regulation of ap modulators. However, Omb affected ap expression neither by directly regulating EGFR signaling, nor via Vg regulation. Therefore, a genetic screen of epigenetic regulators, including the Trithorax group (TrxG) and Polycomb group (PcG) genes was performed. We found that knocking down the TrxG gene kohtalo (kto), domino (dom) or expressing the PcG gene grainy head (grh), the ectopic ap in omb mutants was repressed. The inhibition of apDV by kto knockdown and grh activation could contribute to ap repression. Moreover, Omb and the EGFR pathway are genetically parallel in ap regulation in the ventral compartment. Collectively, Omb is a repressive signal for ap expression in the ventral compartment, which requires TrxG and PcG genes.

Activity of acute pancreatitis is modified by secreted protein acidic and rich in cysteine ablation.

BackgroundAcute pancreatitis (AP) is a frequent cause for hospitalization. However, molecular determinants that modulate severity of experimental pancreatitis are only partially understood.ObjectiveTo investigate the role of secreted protein acidic and rich in cysteine (SPARC) during cerulein‐induced AP in mice.MethodsAP was induced by repeated cerulein injections in SPARC knock‐out mice (SPARC−/−) and control littermates (SPARC+/+). Secreted protein acidic and rich in cysteine expression and severity of AP were determined by histopathological scoring, immunohistochemistry, and biochemical assays. For functional analysis, primary murine acinar cell cultures with subsequent amylase release assays were employed. Proteome profiler assay and ELISA were conducted from pancreatic tissue lysates, and co‐immunofluorescence was performed.ResultsUpon cerulein induction, SPARC expression was robustly induced in pancreatic stellate cells (PSCs) but not in acinar cells. Genetic SPARC ablation resulted in attenuated severity of AP with significantly reduced levels of pancreatic necrosis, apoptosis, immune cell infiltration, and reduced fibrosis upon chronic stimulation. However, the release of amylase upon cerulein stimulation in primary acinar cell culture from SPARC+/+ and SPARC−/− was indistinguishable. Notably, immune cell derived C‐C Motif Chemokine Ligand 2 (CCL2) was highly elevated in SPARC+/+ pancreatic tissue potentially linking PSC derived SPARC with CCL2 induction in AP.ConclusionSPARC mediates the severity of AP. The potential link between SPARC and the CCL2 axis could open new avenues for tailored therapeutic interventions in AP patients and warrants further investigations.

Generation of a nanobody-alkaline phosphatase heptamer fusion for ratiometric fluorescence immunodetection of trace alpha fetoprotein in serum

Alpha fetoprotein (AFP) is an important biomarker for diagnosis of hepatocellular carcinoma (HCC). Whereas, it is always a challenge to detect trace AFP in serum. In this work, a ratiometric fluorescence enzyme immunoassay (RFEIA) was developed using nanobody-alkaline phosphatase (Nb-AP) heptamer and MnFe layered double hydroxides nanoflakes (MnFe LDH) for ultrasensitive detection of AFP. The Nb-AP heptamer (Nb-C4bpα-AP) was constructed by fusion expression of Nb, AP, and α-chain of C4 binding protein (C4bpα), where the C4bpα contributed to multimerization through self-assembly. The dual functional Nb-C4bpα-AP can recognize AFP, dephosphorylate ascorbic acid-2-phosphate (AAP) into ascorbic acid (AA), and thus tune the MnFe LDH-mediated ratiometric fluorescence, which was generated from the oxidization of MnFe LDH on o-phenylenediamine (OPD) and the catalyzation of MnFe LDH on the cyclization reaction between AA and OPD. By integration of Nb-C4bpα-AP, MnFe LDH, AAP, and OPD, the RFEIA showed a limit of detection of 0.013 ng/mL with good selectivity, accuracy and precision. Furthermore, results of clinical serum samples tested by the RFEIA were well confirmed by the automated chemiluminescence immunoassay analyzer. Thus, this work demonstrated that the Nb-C4bpα-AP is a robust immunoreagent and the developed RFEIA could be a very promising tool for diagnosis of HCC.

TLE4 Is a Critical Mediator of Osteoblast and Runx2-Dependent Bone Development.

Healthy bone homeostasis hinges upon a delicate balance and regulation of multiple processes that contribute to bone development and metabolism. While examining hematopoietic regulation by Tle4, we have uncovered a previously unappreciated role of Tle4 on bone calcification using a novel Tle4 null mouse model. Given the significance of osteoblasts in both hematopoiesis and bone development, this study investigated how loss of Tle4 affects osteoblast function. We used dynamic bone formation parameters and microCT to characterize the adverse effects of Tle4 loss on bone development. We further demonstrated loss of Tle4 impacts expression of several key osteoblastogenic genes, including Runx2, Oc, and Ap, pointing toward a potential novel mechanism for Tle4-dependent regulation of mammalian bone development in collaboration with the RUNX family members.

Enhancing Activities of Salt-Tolerant Proteases Secreted by Aspergillus oryzae Using Atmospheric and Room-Temperature Plasma Mutagenesis.

Aspergillus oryzae 3.042 was mutagenized using atmospheric and room-temperature plasma (ARTP) technology to enhance its salt-tolerant proteases activity. Compared to the starting strain, mutant H8 subjected to 180 s of ARTP treatment exhibited excellent genetic stability (15 generations), growth rate, and significantly increased activities of neutral proteases, alkaline proteases, and aspartyl aminopeptidase during fermentation. Mutant H8 significantly enhanced the contents of 1-5 kDa peptides, aspartic acid, serine, threonine, and cysteine in soy sauce by 16.61, 7.69, 17.30, 8.61, and 45.00%, respectively, but it had no effects on the contents of the other 14 free amino acids (FAAs) due to its slightly enhanced acidic proteases activity. Analyses of transcriptional expressions of salt-tolerant alkaline protease gene (AP, gi: 217809) and aspartyl aminopeptidase gene (AAP, gi: 6165646) indicated that their expression levels were increased by approximately 30 and 27%, respectively. But no mutation was found in the sequences of AP and AAP expression cassettes, suggesting that the increased activities of proteases in mutant H8 should be partially attributed to the increased expression of proteases. ARTP technology showed great potential in enhancing the activities of salt-tolerant proteases from A. oryzae.

Immobilization of BMP-2, BMP-7 and alendronic acid on titanium surfaces: Adhesion, proliferation and differentiation of bone marrow-derived stem cells.

This study analyzed the influence of titanium (TiO2 ) surface modifications with two osteogenic proteins (BMP-2, BMP-7) and an anti-osteoclastic drug (alendronic acid [AA]) on sandblasted/acid-etched (SLA) and plain TiO2 (PT) on cell adhesion, proliferation and differentiation (alkaline phosphatase [AP] and osteocalcin [OC]) of bone-marrow derived stem cells (BMSCs) after 1, 3 and 7 days in-vitro. Initially, AA surfaces showed the highest cell number and surface coverage. At day 3 and 7, BMP and AA-modified surfaces exhibited a significantly enhanced cell growth. For proliferation, at days 3 and 7, an enhancement on BMP-2, BMP-7 and AA-surfaces was seen. At day 7, SLA also showed a higher proliferation when compared to PT. Initially, AP expression was elevated on SLA and AA surfaces. At days 3 and 7, a significant increased AP expression was seen for SLA, BMP-2, BMP-7 and AA discs. For OC, SLA and AA surfaces had the highest expression after 1 day whereas after 3 and 7 days a significant difference was recorded for SLA, BMP-2, BMP-7 and AA. In conclusion, a beneficial biological effect of a chemical immobilization method of BMP-2, BMP-7 and alendronate onto titanium surfaces on BMSCs was proven.

Modulation of light-driven arousal by LIM-homeodomain transcription factor Apterous in large PDF-positive lateral neurons of the Drosophila brain.

Apterous (Ap), the best studied LIM-homeodomain transcription factor in Drosophila, cooperates with the cofactor Chip (Chi) to regulate transcription of specific target genes. Although Ap regulates various developmental processes, its function in the adult brain remains unclear. Here, we report that Ap and Chi in the neurons expressing PDF, a neuropeptide, play important roles in proper sleep/wake regulation in adult flies. PDF-expressing neurons consist of two neuronal clusters: small ventral-lateral neurons (s-LNvs) acting as the circadian pacemaker and large ventral-lateral neurons (l-LNvs) regulating light-driven arousal. We identified that Ap localizes to the nuclei of s-LNvs and l-LNvs. In light-dark (LD) cycles, RNAi knockdown or the targeted expression of dominant-negative forms of Ap or Chi in PDF-expressing neurons or l-LNvs promoted arousal. In contrast, in constant darkness, knockdown of Ap in PDF-expressing neurons did not promote arousal, indicating that a reduced Ap function in PDF-expressing neurons promotes light-driven arousal. Furthermore, Ap expression in l-LNvs showed daily rhythms (peaking at midnight), which are generated by a direct light-dependent mechanism rather than by the endogenous clock. These results raise the possibility that the daily oscillation of Ap expression in l-LNvs may contribute to the buffering of light-driven arousal in wild-type flies.

Molecular mapping and characterization of the silkworm apodal mutant.

The morphological diversity of insects is important for their survival; in essence, it results from the differential expression of genes during development of the insect body. The silkworm apodal (ap) mutant has degraded thoracic legs making crawling and eating difficult and the female is sterile, which is an ideal subject for studying the molecular mechanisms of morphogenesis. Here, we confirmed that the infertility of ap female moths is a result of the degradation of the bursa copulatrix. Positional cloning of ap locus and expression analyses reveal that the Bombyx mori sister of odd and bowl (Bmsob) gene is a strong candidate for the ap mutant. The expression of Bmsob is down-regulated, while the corresponding Hox genes are up-regulated in the ap mutant compared to the wild type. Analyses with the dual luciferase assay present a declined activity of the Bmsob promoter in the ap mutant. Furthermore, we demonstrate that Bmsob can inhibit Hox gene expression directly and by suppressing the expression of other genes, including the BmDsp gene. The results of this study are an important contribution to our understanding of the diversification of insect body plan.

Gene-Regulatory Logic to Induce and Maintain a Developmental Compartment.

Gene-Regulatory Logic to Induce and Maintain a Developmental Compartment.

Establishment of a Developmental Compartment Requires Interactions between Three Synergistic Cis-regulatory Modules.

The subdivision of cell populations in compartments is a key event during animal development. In Drosophila, the gene apterous (ap) divides the wing imaginal disc in dorsal vs ventral cell lineages and is required for wing formation. ap function as a dorsal selector gene has been extensively studied. However, the regulation of its expression during wing development is poorly understood. In this study, we analyzed ap transcriptional regulation at the endogenous locus and identified three cis-regulatory modules (CRMs) essential for wing development. Only when the three CRMs are combined, robust ap expression is obtained. In addition, we genetically and molecularly analyzed the trans-factors that regulate these CRMs. Our results propose a three-step mechanism for the cell lineage compartment expression of ap that includes initial activation, positive autoregulation and Trithorax-mediated maintenance through separable CRMs.

Influence of thyroid disorders on the kidney expression and plasma activity of aminopeptidase A.

Thyroid disorders may affect blood pressure and renal function modifying factors of the plasmatic and kidney renin-angiotensin system such as aminopeptidase A (AP A) that metabolizes angiotensin II to angiotensin III. We investigated the expression of AP A in the kidney, as well as its enzymatic activity in the plasma of euthyroid, hyperthyroid, and hypothyroid adult male rats. Hyperthyroidism was induced by daily subcutaneous injections of tetraiodothyronine. Hypothyroid rats were obtained by administration of methimazole in drinking water. Expression of AP A was determined by Western blot analysis. Plasma AP A activity was measured fluorometrically using glutamyl-β-naphthylamide as substrate. While hyperthyroid rats exhibited lower levels of plasma AP A activity than controls, the kidney of hyperthyroid animals expressed significantly higher AP A than controls and hypothyroid animals. A discrepancy between the high expression of AP A in kidney of hyperthyroid rats and the low activity of AP A measured in plasma and kidney of hyperthyroid animals was found. The posttranslational influence of environmental biochemical factors may be in part responsible for that divergence.

Role of the DNA Base Excision Repair Protein, APE1 in Cisplatin, Oxaliplatin, or Carboplatin Induced Sensory Neuropathy

Although chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting side effect of platinum drugs, the mechanisms of this toxicity remain unknown. Previous work in our laboratory suggests that cisplatin-induced CIPN is secondary to DNA damage which is susceptible to base excision repair (BER). To further examine this hypothesis, we studied the effects of cisplatin, oxaliplatin, and carboplatin on cell survival, DNA damage, ROS production, and functional endpoints in rat sensory neurons in culture in the absence or presence of reduced expression of the BER protein AP endonuclease/redox factor-1 (APE1). Using an in situ model of peptidergic sensory neuron function, we examined the effects of the platinum drugs on hind limb capsaicin-evoked vasodilatation. Exposing sensory neurons in culture to the three platinum drugs caused a concentration-dependent increase in apoptosis and cell death, although the concentrations of carboplatin were 10 fold higher than cisplatin. As previously observed with cisplatin, oxaliplatin and carboplatin also increased DNA damage as indicated by an increase in phospho-H2AX and reduced the capsaicin-evoked release of CGRP from neuronal cultures. Both cisplatin and oxaliplatin increased the production of ROS as well as 8-oxoguanine DNA adduct levels, whereas carboplatin did not. Reducing levels of APE1 in neuronal cultures augmented the cisplatin and oxaliplatin induced toxicity, but did not alter the effects of carboplatin. Using an in vivo model, systemic injection of cisplatin (3 mg/kg), oxaliplatin (3 mg/kg), or carboplatin (30 mg/kg) once a week for three weeks caused a decrease in capsaicin-evoked vasodilatation, which was delayed in onset. The effects of cisplatin on capsaicin-evoked vasodilatation were attenuated by chronic administration of E3330, a redox inhibitor of APE1 that serendipitously enhances APE1 DNA repair activity in sensory neurons. These outcomes support the importance of the BER pathway, and particularly APE1, in sensory neuropathy caused by cisplatin and oxaliplatin, but not carboplatin and suggest that augmenting DNA repair could be a therapeutic target for CIPN.

The expression of the aryl hydrocarbon receptor in reproductive and neuroendocrine tissues during the estrous cycle in the pig

The aryl hydrocarbon receptor (AhR) has been recognized as a mediator of xenobiotic-induced toxicity. In addition, it was demonstrated that the AhR is able to influence the regulation of reproductive processes in females. The aim of this study was to examine AhR mRNA (real-time PCR) and protein (Western-blot) expression in ovarian follicles and stroma, corpora lutea (CL), oviducts, endometrium, myometrium as well as in medial basal hypothalami (MBH), and anterior (AP) and posterior (PP) pituitaries harvested during the follicular (days 17–19) and luteal (days 8–10) phase of the porcine estrous cycle. The AhR transcript and protein were found in all structures collected during both phases. AhR mRNA expression tended ( p = 0.06) to be higher in the CL than in follicles. The AhR protein expression in ovarian stroma was higher ( p ≤ 0.01) during the follicular than in the luteal phase. Endometrial expression of AhR mRNA was higher ( p ≤ 0.01), while AhR protein was lower ( p ≤ 0.01) during the follicular phase in comparison to the luteal phase. Within neuroendocrine tissues, AhR mRNA and protein content in hypothalamus were relatively low and did not differ ( p > 0.05) between phases. In contrast, higher AhR mRNA expression in AP ( p ≤ 0.001) and protein expression in PP ( p ≤ 0.01) were found during the luteal phase compared to the follicular phase. Differences in AhR expression observed in reproductive and neuroendocrine tissues during the follicular and luteal phase of the estrous cycle indicate the involvement of AhR in the regulation of reproductive function in pigs.

Evolutionary origin of the insect wing via integration of two developmental modules

Insect wing is a key evolutionary innovation for insect radiation, but its origins and intermediate forms are absent from the fossil record. To understand the ancestral state of the wing, expression of three key regulatory genes in insect wing development, wingless (wg), vestigial (vg), and apterous (ap) was studied in two basal insects, mayfly and bristletail. These basal insects develop dorsal limb branches, tracheal gill and stylus, respectively, that have been considered candidates for wing origin. Here we show that wg and vg are expressed in primordia for tracheal gill and stylus. Those primordia are all located in the lateral body region marked by down-regulation of early segmental wg stripes, but differ in their dorsal-ventral position, indicating their positions drifted within the lateral body region. On the other hand, ap expression was detected in terga of mayfly and bristletail. Notably, the extensive outgrowth of the paranotal lobe of apterygote bristletail developed from the border of ap-expressing tergal margin, and also expressed wg and vg. The data suggest that two regulatory modules involving wg-vg are present in apterygote insects: one associated with lateral body region and induces stick-like dorsal limb branches, the other associated with the boundary of dorsal and lateral body regions and the flat outgrowth of their interface. A combinatorial model is proposed in which dorsal limb branch was incorporated into dorsal-lateral boundary and acquired flat limb morphology through integration of the two wg-vg modules, allowing rapid evolution of the wing.

Intestinal alkaline phosphatase contributes to the reduction of severe intestinal epithelial damage

Inflammatory bowel disease is characterized by chronic inflammation of the intestine and is accompanied by damage of the epithelial lining and by undesired immune responses towards enteric bacteria. It has been demonstrated that intestinal alkaline phosphatase (iAP) protects against the induction of inflammation, possibly due to dephosphorylation of lipopolysaccharide (LPS). The present study investigated the therapeutic potential of iAP in intestinal inflammation and epithelial damage. Intestinal epithelial damage was induced in C57BL/6 mice using detran sulfate sodium (DSS) and iAP was administered 4 days after initial DSS exposure. Loss in body weight was significantly less in iAP-treated mice and accompanied with reduced colon damage (determined by combination of crypt loss, loss of goblet cells, oedema and infiltrations of neutrophils). Treatment with iAP was more effective in case of severe inflammation compared to situations of mild to moderate inflammation. Rectal administration of LPS into a moderate inflamed colon did not aggravate inflammation. Furthermore, soluble iAP did not lower LPS-induced nuclear factor-κB activation in epithelial cells in vitro but induction of cellular AP expression by butyrate resulted in decreased LPS response. In conclusion, the present study shows that oral iAP administration has beneficial effects in situations of severe intestinal epithelial damage, whereas in moderate inflammation endogenous iAP may be sufficient to counteract disease-aggravating effects of LPS. An approach including iAP treatment holds a therapeutic promise in case of severe inflammatory bowel disease.

Regulators Acting in Combinatorial Codes Also Act Independently in Single Differentiating Neurons

In the Drosophila ventral nerve cord, a small number of neurons express the LIM-homeodomain gene apterous (ap). These ap neurons can be subdivided based upon axon pathfinding and their expression of neuropeptidergic markers. ap, the zinc finger gene squeeze, the bHLH gene dimmed, and the BMP pathway are all required for proper specification of these cells. Here, using several ap neuron terminal differentiation markers, we have resolved how each of these factors contributes to ap neuron diversity. We find that these factors interact genetically and biochemically in subtype-specific combinatorial codes to determine certain defining aspects of ap neuron subtype identity. However, we also find that ap, dimmed, and squeeze additionally act independently of one another to specify certain other defining aspects of ap neuron subtype identity. Therefore, within single neurons, we show that single regulators acting in numerous molecular contexts differentially specify multiple subtype-specific traits.

5-azacytidine alters TGF-beta and BMP signaling and induces maturation in articular chondrocytes.

Maintenance of the articular surface depends on the function of articular chondrocytes (ACs) which produce matrix and are constrained from undergoing the maturation program seen in growth plate chondrocytes. Only during pathologic conditions, such as in osteoarthritis, are maturational constraints lost causing recapitulation of the process that occurs during endochondral ossification. With the aim of establishing a model to identify regulatory mechanisms that suppress AC hypertrophy, we examined the capability of 5-azacytidine (Aza) to have an impact on the maturational program of these cells. Primary ACs do not spontaneously express markers of maturation and are refractory to treatment by factors that normally regulate chondrocyte maturation. However, following exposure to Aza, ACs (i) were induced to express type X collagen (colX), Indian hedgehog, and alkaline phosphatase and (ii) showed altered colX and AP expression in response to bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta (TGF-beta), and parathyroid hormone-related protein (PTHrP). Since Aza unmasked responsiveness of ACs to BMP-2 and TGF-beta, we examined the effect of Aza treatment on signaling via these pathways by assessing the expression of the TGF-beta Smads (2 and 3), the BMP-2 Smads (1 and 5), and the Smad2 and 3-degrading ubiquitin E3 ligase Smurf2. Aza-treated ACs displayed less Smad2 and 3 and increased Smad1, 5, and Smurf2 protein and showed a loss of TGF-beta signaling on the P3TP-luciferase reporter. Suggesting that Aza-induction of Smurf2 may be responsible for the loss of Smad2 and 3 protein via this pathway, immunoprecipitation and metabolic labeling experiments confirmed that Aza accelerated the ubiquitination and degradation of these targets. Overall, Aza-treated ACs represent a novel model for the study of mechanisms that regulate maturational potential of articular cartilage, with the data suggesting that maturation of these cells may be due to up-regulation of Smad1 and 5 coupled with a Smurf2-dependent degradation of Smad2 and 3 and loss of TGF-beta signaling.