A conserved site of the prodomain, named the cys residue, coordinates the active zinc site to inhibit catalysis resulting in the protease to remain as a zymogen, known as the cysteine-switch mechanism Loechel et al. Metalloprotease catalysis is dependent on removal of the prodomain by instability of the cysteine switch to expose the active zinc site to enable cleavage of substrates Loechel et al.
The disintegrin domain and cysteine-rich region are also involved in cell—cell interactions. The disintegrin domain binds integrins and the cysteine-rich region supports syndecan-mediated cell adhesion Schlondorff and Blobel, ; Zolkiewska, ; Eto et al. ADAM12 is expressed by adipocytes during adipogenesis Kawaguchi et al.
Intra- and extracellular roles for ADAM12 during adipogenesis in vitro have been proposed: 1 reorganizing the actin cytoskeleton when flat preadipocytes round up, in preparation to lipid-fill into mature adipocytes Kawaguchi et al. Mice overexpressing ADAMS and ADAML showed an accumulation of adipocytes within striated skeletal muscle of the abdominal wall, hind limb, and diaphragm indicative of increased adipogenesis Kawaguchi et al. ADAMS mice lacking the pro- and metalloprotease domain did not display enhanced adipogenesis, suggesting the catalytic metalloprotease domain is crucial for adipogenesis.
This suggests ADAM12 is involved in the development of adipocytes that originate from mesenchymal progenitor or preadipocytes within the perivascular space of connective tissue Kawaguchi et al. ADAM12 gene knockout mice display a significant reduction in the interscapular brown adipose tissue BAT and a mild skeletal muscle defect in the neck and interscapular muscles Kurisaki et al. A study by Masaki et al. HB-EGF was involved in this phenotype but rather inhibited adipogenesis, questioning a role for ADAM12 ectodomain sheddase activity in promoting adipogenesis as suggested by Kurisaki et al.
ADAM12 has shown that it is an important regulator of fat development. However, the exact role ADAM12 has during adipocyte proliferation and differentiation is still unclear. This investigation aims to determine the molecular pathways ADAM12 influences during proliferation and differentiation of adipocytes.
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Phase images demonstrate the morphological change during adipocyte differentiation from flattened preadipocytes day 6 to mature lipid-filled adipocytes day 9—day 13; Figure 1. The mean diameter of lipid droplets contained within mature adipocytes was found to be different between the control and ADAM12 RNAi cells. These findings suggest ADAM12 is involved in differentiation of fibroblastic-like preadipocytes into round adipocytes and development of mature lipid-filled adipocytes. To rule out the possibility that increased apoptosis was driving this effect, we evaluated the expression of Bcl2 and Bax , two key regulators of apoptosis.
At the early stages of ADAM12 knockdown day 3 , expression of Bcl2 transcript was increased; there was no difference compared with controls at days 6 and 9. G3PDH glycerolphosphate dehydrogenase enzyme activity was used as an early marker of adipocyte differentiation in response to ADAM12 gene knockdown. Its activity is correlated to the early stages of glucose metabolism lipogenesis during adipogenesis. The effect of ADAM12 knockdown on fatty-acid synthesis during latter stages of adipogenesis was assayed by measuring fatty-acid synthase FAS activity.
This relates to lipid filling triglyceride anabolism in maturing adipocytes.
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These results, supported by an increase in the size of lipid droplets at day 13 Figure 2 , suggests there was increased lipogenesis in ADAM12 RNAi cells during stages where adipocytes were lipid-filling. Multidimensional scaling analysis of the filtered and normalized microarray data showed that time was the biggest factor influencing the data, with day 3 T3 , day 6 T6 , and day 9 T9 samples all showing separation in the first principal component Figure 4A.
A control sample at T3 and ADAM12 RNAi sample at T9 were outliers from their respective groups in the second principal component; however, they did cluster with their corresponding time points in the first principal component Figure 4A. With the outlying samples removed, the remaining samples cluster tightly within their time points in the first two principal components Figure 4B. Quality control for microarray gene profiling. A PC1 vs. PC2 shows the greatest effect is time.
Two outliers are evident for T3 and T9. B PC1 vs. PC2 with outliers removed for T3 and T9. Samples cluster within time. C PC1 vs. PC4 T3 and T9 outliers removed shows clear separation between treatment groups control and treated. Note: Gene expression was background corrected using the normexp method and then normalized using quantile normalization.
The biggest impact of ADAM12 gene knockdown on the number of differentially expressed genes was at T3, followed by T6 and T9, respectively. Values in overlap represent the number of genes found to be different in more than one comparison.
Although ADAM12 gene expression returned to control levels in T9, substantial differences are still evident at T9, both at the transcript level and at the protein level, based on the phenotype observed in vitro. We believe that these differences are attributed to changes occurring early in the time course when cells are proliferating and differentiating; in particular, changes in the extracellular matrix which are crucial for adipogenesis.dersmaroremo.tk
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Gene lists for DE genes for each time comparison can be found in Supplemental File 1. The samples generally cluster both by time point and treatment, for all comparisons. For the ALL comparison, the difference between the ADAM12 RNAi and control is obvious at all three time points; however, the overall level expression for many of the genes does tend to change substantially over time Figure 6A.
For the comparisons at individual time points, as expected, the difference beween treatment and control is most apparent at the time point tested, and tends to be less visible at the other time points Figure 6, B—D.
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This indicates that these are likely to be the genes most directly impacted by the ADAM12 knockdown. The top 10 GO terms for each comparison can be seen in Figure 7. We then wanted to determine what genes were involved in causing these changes. Table 1 outlines the top 10 core-matrisome proteins differentially expressed top five up-regulated and five down-regulated with knockdown of ADAM12; refer to Supplemental File 3 for an extensive list.
Ctgf connective tissue growth factor was the most highly up-regulated matrisome gene for ALL 1. Collagens are important structural ECM proteins fundamental to support the dramatic morphological changes in the adipocytes during differentiation. Proteins associated with collagen binding and synthesis were also found to be the greatest up-regulated core-matrisome genes; these included Dcn decorin; ALL, 0. Core-matrisome genes Naba et al. T3: day 3 in culture posttransfection; T6: day 6 in culture posttransfection; T9: day 9 in culture posttransfection; ALL: across all three time points T3, T6, and T9.
Magnitude of regulation is based on fold change. Note: These data are from a trial independent to the microarray. In addition to core-matrisome proteins, we were also interested in proteins associated with the matrisome, such as cytokines, chemokines, growth factors, mucins, galectins, plexins, serine proteases, matrix metalloproteases, and other ECM-associated proteins. MMP3 was in the 20 top-ranked genes for all comparisons.
The effect of ADAM12 gene knockdown on gene expression of matrisome genes. Values in the overlap represent the number of genes found to be different in more than one comparison. Mmp3 is top ranked for all comparisons. Mmp3 : matrix metalloprotease 3; Mfab5 : microfibrillar-associated protein 5; Fbn1 : fibrillin 1; Ctgf : connective tissue growth factor.
The top 10 canonical pathways for the ALL comparison and the six top-ranked sets for T3, T6, and T9 comparisons are summarized in Table 2 ; refer to Supplemental File 5 for the full lists. The same pathway was ranked second at T6 and was also the top-ranked pathway at T9, suggesting the effects of ADAM12 gene knockdown were maintained even though there was little difference in the gene expression of ADAM12 at T9. Gene expression of Igfbp3 T3, 0.
The metabolic pathways found to be differentially regulated that may contribute to altered G3PDH and FAS activity were PPAR signaling, mitochondrial fatty-acid beta oxidation, glucose metabolism, and fatty-acid metabolism. Several pathways related to the ECM were up-regulated in ADAM12 RNAi cells at multiple time points: degradation of the extracellular matrix; keratan sulfate degradation for the ALL comparison; and keratan sulfate degradation, keratan sulfate keratin metabolism, and extracellular matrix organization at T9.
Other immune-related pathways were chemokine receptors bind chemokines ALL, T9 , graft versus host disease T6 , and allograft rejection T6. Based on the results of our analysis, it appears that ADAM12 is either directly or indirectly influencing numerous pathways during adipogenesis. In addition, IGFBP3 can regulate the transcription of adiponectin, an adipocytokine important for fatty-acid oxidation through activation of AMPK adenosine monophosphate-activated protein kinase.
We wanted to use an established model of adiponenesis in vitro to investigate this further.
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Using metabolic markers of adipocyte differentiation and gene profiling we were able to more clearly define pathways involved in regulation of adipogenesis and identify novel pathways for ADAM12 during adipogenesis. Following clonal expansion, the cells arrest and differentiate into a round adipocyte. Following the early stage of differentiation, cells begin lipid-filling to become mature adipocytes. The primary role of this mature adipocyte is to store energy and break down fatty acids to provide ATP when available systemic sources become low. During the course of development the ECM serves as a structure to support the large changes in cell morphology and to harvest and sequester ligands important for signaling proliferation and differentiation.
In turn, this potentially initiates a feedback loop to increase expression of IGFBPs and other molecules in the pathway responsible for cleavage of IGFBPs to cause it to be up-regulated. Collectively these data suggest a role for ADAM12 in proliferation during adipogenesis. This impacts downstream signaling of the mTOR signaling pathway impeding proliferation of preadipocytes. Working model for ADAM12 during proliferation in preadipocytes.
Three of the top five signaling pathways differentially regulated when preadipocytes were proliferating and knockdown of ADAM12 gene expression was the greatest day 3 were related to IFN interferon signaling, interferon alpha beta signaling, interferon gamma signaling, and interferon signaling. It is not clear from this study whether suppression of IFN signaling is in response to impeded cell growth as a mechanism to maintain proliferation or if ADAM12 directly impacts IFN signaling by remodeling of the ECM or via direct interaction.
Following proliferation and growth arrest of preadipocytes, we investigated the effect of ADAM12 knockdown on enzymatic markers of differentiation. We also measured when preadipocytes differentiated to round adipocytes, the size of mature lipid-filled adipocytes, and the size of large lipid droplets in mature adipocytes. We found on day 9 that the number of preadipocytes was greater in ADAM12 RNAi cells and there were less adipocytes, suggesting differentiation was delayed.
On day 13, the size of adipocytes was not different in ADAM12 RNAi cells but the size of lipids in cells with one, two, and three lipid droplets was found to be greater. Kawaguchi et al. Reorganization of the fibronectin-rich matrix during preadipocytes differentiation is one explanation for ADAM12 involvement in this as it has been shown to cleave fibronectin Roy et al.
Up-regulation of Fbn1 was found in the adipose tissue of obese women and associated with increased adipocyte size Davis et al. A role for IGFBP3 is also possible as it has been implicated in inhibition of preadipocyte differentiation and further development of adipocyte differentiation Baxter and Twigg, We found the mitochondrial fatty-acid beta oxidation pathway ALL gene list to be down-regulated.
Linked to this, adipocytokine signaling pathway and activated AMPK stimulates fatty-acid oxidation were also down-regulated ALL gene list. Adiponectin is an energy sensing cytokine which stimulates AMPK to increase fatty-acid oxidation, reducing the triglyceride content of adipocytes in adipose tissue and skeletal muscle when energy is low. Reduced expression of adiponectin and another adipocytokine, Cfd adipsin , is associated with hypertrophy of adipocytes in obesity and insulin resistance in obesity and type II diabetes Rosen et al. Further studies are required to determine what is driving increased lipogenesis and reduced signaling of fatty-acid oxidation pathways as a result of ADAM12 gene knockdown.
Working model for ADAM12 during differentiation of preadipocytes and maturation of adipocytes. In the presence of ADAM12, PPARy—the master regulator of adipogenesis—stimulates genes for adipocytes to differentiate changing their morphology from a flattened preadipocyte to a round cell.
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