SummaryAxon specification is morphologically reproducible in vitro, whereas dendrite formation differs in vitro and in vivo. Cortical neurons initially develop immature neurites, but in vivo these are eliminated concurrently with the formation of a leading process, the future dendrite. However, the molecular mechanisms underlying these neuronal maturation events remain unclear. Here we show that caveolin-1, a major component of caveolae that are never observed in neurons, regulates in vivo-specific steps of neuronal maturation. Caveolin-1 is predominantly expressed in immature cortical neurons and regulates clathrin-independent endocytosis. In vivo knockdown of caveolin-1 disturbs immature neurite pruning, leading process elongation, and subsequent neuronal migration. Importantly, N-cadherin and L1, which are required for immature neurite formation, undergo caveolin-1-mediated endocytosis to eliminate immature neurites. Collectively, our findings indicate that caveolin-1 regulates N-cadherin and L1 trafficking independent of caveolae, which contributes to spatiotemporally restricted cellular events; immature neurite pruning and leading process elongation during early neuronal maturation.

Coxsackie virus and adenovirus receptor-like membrane protein (CLMP) was identified as the tight junctionassociated transmembrane protein of epithelial cells with homophilic binding activities. CLMP is also recognized as adipocyte adhesion molecule (ACAM), and it is upregulated in mature adipocytes in rodents and humans with obesity. Here, we present that aP2 promoter-driven ACAM transgenic mice are protected from obesity and diabetes with the prominent reduction of adipose tissue mass and smaller size of adipocytes. ACAM is abundantly expressed on plasma membrane of mature adipocytes and associated with formation of phalloidin-positive polymerized form of cortical actin (F-actin). By electron microscopy, the structure of zonula adherens with an intercellular space of ∼10-20 nm was observed with strict parallelism of the adjoining cell membranes over distances of 1-20 mm, where ACAM and g-actin are abundantly expressed. The formation of zonula adherens may increase the mechanical strength, inhibit the adipocyte hypertrophy, and improve the insulin sensitivity.Quite a few adhesion molecules have been identified by molecular genetics as well as gene expression profile studies in adipocytes derived from experimental models and human studies. For instance, only cadherins were reported to be expressed in premature adipocytes. In the cell lines, such as C3H10T1/2 and 3T3-L1 cells, N-cadherin and cadherin-11 are expressed, and they are prominently suppressed by the induction of adipocyte differentiation and downregulated to very low levels after the full differentiation (1). Transgenic (Tg) expression of dominant-negative N-cadherin decreased bone formation, delayed acquisition of peak bone mass, and increased body fat (2). Although the information of adhesion molecules in adipocyte biology is limited, we identified adipocyte adhesion molecule (ACAM) from the visceral adipose tissues of OLETF (Otsuka Long-Evans Tokushima fatty) rats by PCR-based cDNA suppressive subtraction methods (3). Mouse ACAM was independently identified as adipocyte-specific protein 5 (ASP5) from 3T3-L1 cells by using signal sequence trap by a retrovirus-mediated expression screening method (4). Human ACAM had been identified as coxsackie virus and adenovirus receptor-like membrane protein (CLMP) by bioinformatics approaches, and Raschperger et al. (5) demonstrated that CLMP is a component of the tight junction of epithelial cells and colocalized with zonula occludens-1. ACAM/CLMP belongs to CTX (cortical thymocyte marker in Xenopus), and they are characterized by extracellular variable (V-type) and constant (C2-type) immunoglobulin domains, which are involved in the homophilic adhesion and aggregation of the cells. Although we reported the expression of ACAM increased in mature adipocytes in genetically obese db/db and diet-induced obesity mice, and also in adipose tissues

A disintegrin and metalloproteinase 12 (ADAM12) is known to be involved in chondrocyte proliferation and maturation however the mechanisms are not fully understood. In this study expression and localization of ADAM12 during chondrocyte differentiation was examined in the mouse growth plate by immunohistochemistry. Adam12 expression during ATDC5 chondrogenic differentiation was examined by real-time PCR and compared with the expression pattern of type X collagen. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system was used to generate Adam12-knockout (KO) ATDC5 cells. Adam12-KO and Adam12 overexpressing cells were used for analyses of ADAM12 expression with or without TGF-β1 stimulation. ADAM12 was identified predominantly in chondrocytes of the proliferative zone in mouse growth plates by immunohistochemistry. Adam12 was upregulated prior to Col10a1 during chondrogenic differentiation in wildtype ATDC5 cells. In Adam12-KO ATDC5 cells, following initiation of chondrogenic differentiation, we observed a reduction in Igf-1 expression along with an upregulation of hypertrophy associated Runx2, Col10a1, and type X collagen protein expression. In ATDC5 wild-type cells, stimulation with TGF-β1 upregulated the expression of Adam12 and Igf-1 and downregulated the expression of Runx2. In contrast, in Adam12-KO ATDC5 cells, these TGF-β1-induced changes were suppressed. Adam12 overexpression resulted in an upregulation of Igf-1 and downregulation of Runx2 expression in ATDC5 cells. The findings suggest that ADAM12 is important in the 2 regulation of chondrocyte differentiation, potentially by regulation of TGF-β1 dependent signaling and that targeting of ADAM12 may have a role in management of abnormal chondrocyte differentiation.