Different ADAMs have distinct influences on Kit ligand processing: phorbol-ester-stimulated ectodomain shedding of Kitl1 by ADAM17 is reduced by ADAM19.

Kit ligand (Kitl), the ligand for the Kit receptor tyrosine kinase, plays important roles in hematopoiesis, gametogenesis and melanogenesis. Kitl is synthesized as a membrane-anchored precursor that can be processed to produce the soluble growth factor. Here, we evaluated the role of ADAM (a disintegrin and metalloprotease) metalloproteases in ectodomain shedding of Kitl. We found that both ADAM17 and ADAM19 affect Kitl1 shedding, albeit in different ways. Overexpression of ADAM19 resulted in decreased levels of Endo-H-resistant mature Kitl1, thereby reducing the amount of Kitl that is shed from cells following stimulation with phorbol esters. ADAM17 was identified as the major phorbol-ester-stimulated sheddase of Kitl1, whereas ADAMs 8, 9, 10, 12 and 15 were not required for this process. ADAM17 also emerged as the major constitutive and phorbol-ester-stimulated sheddase of Kitl2 in mouse embryonic fibroblasts. Mutagenesis of the juxtamembrane domain of Kitl2 showed no stringent sequence requirement for cleavage by ADAM17, although two nonadjacent stretches of four amino acid residues were identified that are required for Kitl2 shedding. Taken together, this study identifies a novel sheddase, ADAM17, for Kitl1 and Kitl2, and demonstrates that ADAM19 can reduce ADAM17-dependent phorbol-ester-stimulated Kitl1 ectodomain shedding.

Compensation for dystrophin-deficiency: ADAM12 overexpression in skeletal muscle results in increased alpha 7 integrin, utrophin and associated glycoproteins.

Mouse models for genetic diseases are among the most powerful tools available for developing and testing new treatment strategies. ADAM12 is a disintegrin and metalloprotease, previously demonstrated to significantly alleviate the pathology of mdx mice, a model for Duchenne muscular dystrophy in humans. More specifically ADAM12 appeared to prevent muscle cell necrosis in the mdx mice as evidenced by morphological analysis and by the reduced levels of serum creatine kinase. In the present study we demonstrated that ADAM12 may compensate for the dystrophin deficiency in mdx mice by increasing the expression and redistribution of several components of the muscle cell-adhesion complexes. First, we analyzed transgenic mice that overexpress ADAM12 and found mild myopathic changes and accelerated regeneration following acute injury. We then analyzed changes in gene-expression profiles in mdx/ADAM12 transgenic mice compared with their littermate controls and found only a few genes with an expression change greater than 2-fold between mdx/ADAM12 and mdx. The small changes in gene expression were unexpected, considering the marked improvement of the mdx pathology when ADAM12 is overexpressed, and suggested that significant changes in mdx/ADAM12 muscle might occur post-transcriptionally. Indeed, by immunostaining and immunoblotting we found an approximately 2-fold increase in expression, and distinct extrasynaptic localization, of alpha 7B integrin and utrophin, the functional homolog of dystrophin. The expression of the dystrophin-associated glycoproteins was also increased. In conclusion, these results demonstrate a novel way to alleviate dystrophin deficiency in mice, and may stimulate the development of new approaches to compensate for dystrophin deficiency in animals and humans.

ADAM12 induces actin cytoskeleton and extracellular matrix reorganization during early adipocyte differentiation by regulating beta1 integrin function.

Changes in cell shape are a morphological hallmark of differentiation. In this study we report that the expression of ADAM12, a disintegrin and metalloprotease, dramatically affects cell morphology in preadipocytes, changing them from a flattened, fibroblastic appearance to a more rounded shape. We showed that the highest levels of ADAM12 mRNA were detected in preadipocytes at the critical stage when preadipocytes become permissive for adipogenic differentiation. Furthermore, as assessed by immunostaining, ADAM12 was transiently expressed at the cell surface concomitant with the reduced activity of beta1 integrin. Co-immunoprecipitation studies indicated the formation of ADAM12/beta1 integrin complexes in these preadipocytes. Overexpression of ADAM12 at the cell surface of 3T3-L1 preadipocytes achieved by transient transfection or retroviral transduction led to the disappearance of the extensive network of actin stress fibers that are characteristic of these cells, and its reorganization into a cortical network located beneath the cell membrane. The cells became more rounded, exhibited fewer vinculin-positive focal adhesions, and adhered less efficiently to fibronectin in attachment assays. Moreover, ADAM12-expressing cells were more prone to apoptosis, which could be prevented by treating the cells with beta1-activating antibodies. A reduced and re-organized fibronectin-rich extracellular matrix accompanied these changes. In addition, beta1 integrin was more readily extracted with Triton X-100 from cells overexpressing ADAM12 than from control cells. Collectively, these results show that surface expression of ADAM12 impairs the function of beta1 integrins and, consequently, alters the organization of the actin cytoskeleton and extracellular matrix. These events may be necessary for early adipocyte differentiation.

ADAM12 alleviates the skeletal muscle pathology in mdx dystrophic mice.

Muscular dystrophy is characterized by muscle degeneration and insufficient regeneration and replacement of muscle fibers by connective tissue. New therapeutic strategies directed toward various forms of muscular dystrophy are needed to preserve muscle mass and promote regeneration. In this study we examined the role of the transmembrane ADAM12, a disintegrin and metalloprotease, which is normally associated with development and regeneration of skeletal muscle. We demonstrate that ADAM12 overexpression in the dystrophin-deficient mdx mice alleviated the muscle pathology in these animals, as evidenced by less muscle cell necrosis and inflammation, lower levels of serum creatine kinase, and less uptake of Evans Blue dye into muscle fibers. These studies demonstrate that ADAM12 directly or indirectly contributes to muscle cell regeneration, stability, and survival.

ADAM 12 protease induces adipogenesis in transgenic mice.

ADAM 12 (meltrin-alpha) is a member of the ADAM (a disintegrin and metalloprotease) family. ADAM 12 functions as an active metalloprotease, supports cell adhesion, and has been implicated in myoblast differentiation and fusion. Human ADAM 12 exists in two forms: the prototype membrane-anchored protein, ADAM 12-L, and a shorter secreted form, ADAM 12-S. Here we report the occurrence of adipocytes in the skeletal muscle of transgenic mice in which overexpression of either form is driven by the muscle creatine kinase promoter. Cells expressing a marker of early adipogenesis were apparent in the perivascular space in muscle tissue of 1- to 2-week-old transgenic mice whereas mature lipid-laden adipocytes were seen at 3 to 4 weeks. Moreover, female transgenics expressing ADAM 12-S exhibited increases in body weight, total body fat mass, abdominal fat mass, and herniation, but were normoglycemic and did not exhibit increased serum insulin, cholesterol, or triglycerides. Male transgenics were slightly overweight and also developed herniation but did not become obese. Transgenic mice expressing a truncated form of ADAM 12-S lacking the prodomain and the metalloprotease domain did not develop this adipogenic phenotype, suggesting a requirement for ADAM 12 protease activity. This is the first in vivo demonstration that an ADAM protease is involved in adipogenesis.

Endogenous adipocyte precursor cells for regenerative soft-tissue engineering.

Subcutaneous injection of reconstituted basement membrane (Matrigel) in combination with basic fibroblast growth factor induces de novo adipogenesis in which endogenous precursor cells invade the artificially formed Matrigel space, proliferate and differentiate to form adipose tissue. Since this adipogenesis offers us a novel approach for soft-tissue reconstruction without transplanting preadipocytes, the early process was examined by optical and electron microscopy. Formation of multiple layers of fibroblast-like cells at the surface of Matrigel implant was the first response of connective tissue. The cells within four to five layers proximal to Matrigel implant acquired a thick cytoplasm and an enlarged nucleus, and they invaded Matrigel space together with endothelial cells which caused neovascularization. Phagocytotic incorporation and digestion of Matrigel components by well-developed lysosomes appeared to be a stimulus of fibroblast-like cells to mature depending on proximity to Matrigel. The fibroblast-like cells often contacted to the outer surface of capillary over a large area and rapidly accumulated lipid droplets. Electron microscopy of the developing adipocytes showed a well-organized smooth endoplasmic reticulum and mitochondria. This investigation thus revealed the characteristics of adipocyte precursor cells, which can be recruited for regenerative engineering of soft tissues.