Uterine leiomyomas (ULs) are benign tumors occurring in the majority of reproductive aged women. Despite the high prevalence of these tumors, little is known about their etiology. A hallmark of ULs is the excessive deposition of extracellular matrix (ECM), primarily collagens. Collagens are known to modulate cell behavior and function singularly or through interactions with integrins and growth factor-mediated mitogenic pathways. To better understand the pathogenesis of ULs and the role of ECM collagens in their growth, we investigated the interaction of leiomyoma smooth muscle cells (LSMCs) with two different forms of collagen, non-polymerized collagen (monomeric) and polymerized collagen (fibrillar), in the absence or presence of platelet-derived growth factor (PDGF), an abundant growth factor in ULs. Primary cultures of human LSMCS from symptomatic patients were grown on these two different collagen matrices and their morphology, cytoskeletal organization, cellular proliferation, and signaling pathways were evaluated. Our results showed that LSMCs had distinct morphologies on the different collagen matrices and their basal as well as PDGF-stimulated proliferation varied on these matrices. These differences in proliferation were accompanied by changes in cell cycle progression and p21, an inhibitory cell cycle protein. In addition we found alterations in the phosphorylation of focal adhesion kinase, cytoskeletal reorganization, and activation of the mitogen activated protein kinase (MAPK) signaling pathway. In conclusion, our results demonstrate a direct effect of ECM on the proliferation of LSMCs through interplay between the collagen matrix and the PDGF-stimulated MAPK pathway. In addition, these findings will pave the way for identifying novel therapeutic approaches for ULs that target ECM proteins and their signaling pathways in ULs.

Basigin (BSG) is a multifunctional glycoprotein that plays an important role in male reproduction since male knockout (KO) mice are sterile. The Bsg KO testis lacks elongated spermatids and mature spermatozoa, a phenotype similar to that of alpha-mannosidase IIx (MX) KO mice. MX regulates formation of N-acetylglucosamine (GlcNAc) terminated N-glycans that participate in germ cell-Sertoli cell adhesion. Results showed that Bsg KO spermatocytes displayed normal homologous chromosome synapsis and progression through meiosis. However, only punctate expression of the round spermatid marker SP-10 in the acrosomal granule of germ cells of Bsg KO mice was detected indicating that spermatogenesis in Bsg KO mice was arrested at the early round spermatid stages. We observed a large increase in the number of germ cells undergoing apoptosis in Bsg KO testes. Using lectin blotting, we determined that GlcNAc terminated N-glycans are linked to BSG. GlcNAc terminated N-glycans were significantly reduced in Bsg KO testes. These observations indicate that BSG may act as a germ cell-Sertoli cell attachment molecule. Loss of BSG significantly reduced adhesion between GC-2 and SF7 cells. Moreover, wild type testes showed strong expression of N-cadherin (CDH2) while expression was greatly reduced in the testes of Bsg KO mice. In addition, the integrity of the blood-testis barrier (BTB) was compromised in Bsg KO testes. In conclusion, although some Bsg KO spermatogonia can undergo normal progression to the spermatocyte stage, BSG-mediated germ cell-Sertoli cell interactions appear to be necessary for integrity of the BTB and spermatocyte progression to mature spermatozoa.

Tumor cell migration is a crucial step in the metastatic cascade, and interruption of this step is considered to be logically effective in preventing tumor metastasis. Lipid rafts, distinct liquid ordered plasma membrane microdomains, have been shown to influence cancer cell migration, but the underlying mechanisms are still not well understood. Here, we report that lipid rafts regulate the dynamics of actin cytoskeleton and focal adhesion in human melanoma cell migration. Disrupting the integrity of lipid rafts with methyl-β cyclodextrin enhances actin stress fiber formation and inhibits focal adhesion disassembly, accompanied with alterations in cell morphology. Furthermore, actin cytoskeleton, rather than microtubules, mediates the lipid raft-dependent focal adhesion disassembly by regulating the dephosphorylation of focal adhesion proteins and the internalization of β3 integrin. We also show that Src-RhoA-Rho kinase signaling pathway is responsible for lipid raft disruption-induced stress fiber formation. Taken together, these observations provide a new mechanism to further explain how lipid rafts regulate the migration of melanoma cell and suggest that lipid rafts may be novel and attractive targets for cancer therapy.

The neural cell adhesion molecule (NCAM) plays critical roles in multiple cellular processes in neural cells, mesenchymal stem cells, and various cancer cells. However, the effect and mechanism of NCAM in human melanoma cells are still unclear. In this study, we found that NCAM regulated the proliferation, apoptosis, autophagy, migration, and epithelial‐to‐mesenchymal transition of human melanoma cells by determining the biological behavior of NCAM knockdown A375 and M102 human melanoma cells. Further studies revealed that NCAM knockdown impaired the organization of actin cytoskeleton and reduced the phosphorylation of cofilin, an actin‐cleaving protein. When cells were transfected with cofilin S3A (dephosphorylated cofilin), biological behavior similar to that of NCAM knockdown cells was observed. Research on the underlying molecular mechanism showed that NCAM knockdown suppressed activation of the Src/Akt/mTOR pathway. Specific inhibitors of Src and PI3K/Akt were employed to further verify the relationship between Src/Akt/mTOR signaling and cofilin, and the results showed that the phosphorylation level of cofilin decreased following inhibition of the Src/Akt/mTOR pathway. These results indicated that NCAM may regulate the proliferation, apoptosis, autophagy, migration, and epithelial‐to‐mesenchymal transition of human melanoma cells via the Src/Akt/mTOR/cofilin pathway‐mediated dynamics of actin cytoskeleton.

S U M M A R Y b-catenin plays a role in cell adhesion and as a transcriptional coactivator. Its levels are regulated in cells by controlling its degradation through ubiquitination by two different E3 ligase complexes. One complex contains b-transducing repeat containing (BTRC) protein, which binds to b-catenin when phosphorylated on specific (S33 and S37) residues, whereas the other involves calcyclin-binding protein (CACYBP). The aim of this study was to determine the localization and levels of total and active (S33/S37-dephosphorylated) b-catenin in the pregnant mouse uteri and those undergoing artificially stimulated decidualization. These two forms of b-catenin were localized almost exclusively to the endometrial epithelia just prior to the onset of implantation. Although this localization continued after the onset of implantation, there were less epithelial cells present in areas of the uterus undergoing decidualization. Rather, there was a progressive increase in b-catenin localization in endometrial stromal cells undergoing decidualization in the anti-mesometrial and, to a lesser extent, in the mesometrial regions. The presence of a conceptus was not required for the changes in localization seen in the pregnant uterus because similar findings were also seen in uteri undergoing artificially stimulated decidualization. Finally, overall levels of total, active (S33 and S37 dephosphorylated), and phosphorylated (S33/S37/T42) b-catenin protein and the steady-state levels of calcyclin-binding protein mRNA changed in the uterus during decidualization. The result of this study shows the changing localization and levels of b-catenin in the mouse uterus during decidualization. Further, the results suggest potential roles for both the BTRC and CACYBP E3 ligase mechanisms of b-catenin ubiquitination in the uterus during decidualization. (J Histochem Cytochem 55:963-974, 2007)