Integrin Trafficking and Tumor Progression

Integrins are major mediators of cancer cell adhesion to extracellular matrix. Through this interaction, integrins play critical roles in cell migration, invasion, metastasis, and resistance to apoptosis during tumor progression. Recent studies highlight the importance of integrin trafficking, endocytosis and recycling, for the functions of integrins in cancer cells. Understanding the molecular mechanisms of integrin trafficking is pivotal for understanding tumor progression and for the development of anticancer drugs

Exosomes from Human Adipose Tissue-Derived Mesenchymal Stem Cells Promote Epidermal Barrier Repair by Inducing de Novo Synthesis of Ceramides in Atopic Dermatitis.

Atopic dermatitis (AD) is a multifactorial, heterogeneous disease associated with epidermal barrier disruption and intense systemic inflammation. Previously, we showed that exosomes derived from human adipose tissue-derived mesenchymal stem cells (ASC-exosomes) attenuate AD-like symptoms by reducing multiple inflammatory cytokine levels. Here, we investigated ASC-exosomes' effects on skin barrier restoration by analyzing protein and lipid contents. We found that subcutaneous injection of ASC-exosomes in an oxazolone-induced dermatitis model remarkably reduced trans-epidermal water loss, while enhancing stratum corneum (SC) hydration and markedly decreasing the levels of inflammatory cytokines such as IL-4, IL-5, IL-13, TNF-α, IFN-γ, IL-17, and TSLP, all in a dose-dependent manner. Interestingly, ASC-exosomes induced the production of ceramides and dihydroceramides. Electron microscopic analysis revealed enhanced epidermal lamellar bodies and formation of lamellar layer at the interface of the SC and stratum granulosum with ASC-exosomes treatment. Deep RNA sequencing analysis of skin lesions demonstrated that ASC-exosomes restores the expression of genes involved in skin barrier, lipid metabolism, cell cycle, and inflammatory response in the diseased area. Collectively, our results suggest that ASC-exosomes effectively restore epidermal barrier functions in AD by facilitating the de novo synthesis of ceramides, resulting in a promising cell-free therapeutic option for treating AD

Contribution of a Low-Barrier Hydrogen Bond to Catalysis Is Not Significant in Ketosteroid Isomerase

Low-barrier hydrogen bonds (LBHBs) have been proposed to have important influences on the enormous reaction rate increases achieved by many enzymes. Δ5-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Δ5-3-ketosteroid to its conjugated Δ4-isomers at a rate that approaches the diffusion limit. Tyr14, a catalytic residue of KSI, has been hypothesized to form an LBHB with the oxyanion of a dienolate steroid intermediate generated during the catalysis. The unusual chemical shift of a proton at 16.8 ppm in the nuclear magnetic resonance spectrum has been attributed to an LBHB between Tyr14 Oη and C3-O of equilenin, an intermediate analogue, in the active site of D38N KSI. This shift in the spectrum was not observed in Y30F/Y55F/D38N and Y30F/Y55F/Y115F/D38N mutant KSIs when each mutant was complexed with equilenin, suggesting that Tyr14 could not form LBHB with the intermediate analogue in these mutant KSIs. The crystal structure of Y30F/Y55F/Y115F/D38N-equilenin complex revealed that the distance between Tyr14 Oη and C3-O of the bound steroid was within a direct hydrogen bond. The conversion of LBHB to an ordinary hydrogen bond in the mutant KSI reduced the binding affinity for the steroid inhibitors by a factor of 8.1–11. In addition, the absence of LBHB reduced the catalytic activity by only a factor of 1.7–2. These results suggest that the amount of stabilization energy of the reaction intermediate provided by LBHB is small compared with that provided by an ordinary hydrogen bond in KSI

Hypoxia stimulates carcinoma invasion by stabilizing microtubules and promoting the Rab11 trafficking of the alpha6beta4 integrin

Hypoxia plays a key role in tumor cell survival, invasion, and metastasis. Here we show that hypoxia increases tumor cell invasion by the modulation of Rab11, an important molecule for vesicular trafficking, especially membrane protein recycling and translocation of proteins from trans-Golgi network to plasma membrane. Dominant-negative Rab11 dramatically decreased hypoxia-induced invasion of MDA-MB-231 breast carcinoma cells without affecting cell apoptosis. Hypoxia-induced Rab11 trafficking is regulated by microtubule stability, as evidenced by the findings that hypoxia increases Glu tubulin and that colchicine blocks Rab11 trafficking and invasion. Inhibition of GSK-3beta activity by hypoxia seems to be central to microtubule stabilization and invasion. In fact, expression of a dominant-negative GSK-3beta was sufficient to stimulate invasion in normoxia. One target of Rab11-mediated trafficking that contributes to invasion is the integrin alpha6beta4. Hypoxia induced a significant increase in alpha6beta4 surface expression but it had no effect on the surface expression of alpha3beta1. This increase is dependent on Rab11 and stable microtubules. In summary, we identify vesicle trafficking as a novel target of hypoxic stimulation that is important for tumor invasion

Ras stimulation of E2F activity and a consequent E2F regulation of integrin alpha6beta4 promote the invasion of breast carcinoma cells

Active Ras proteins contribute to breast carcinogenesis and progression. Here, we provide evidence that active H-Ras regulates the expression and activity of the E2F family of transcription factors in SUM-159 breast carcinoma cells. In addition, we show by using a DNA-binding mutant of E2F, as well as expression of specific E2Fs that are transcriptionally active, that the active E2Fs1-3 can mediate the H-Ras-dependent invasion of SUM-159 cells. The inhibitory E2Fs4-5, in contrast, do not influence invasion. One mechanism by which the active E2Fs promote H-Ras-dependent invasion seems to be their ability to increase expression of the beta4 integrin subunit, a component of the alpha6beta4 integrin that is known to enhance carcinoma invasion. Specifically, expression of E2Fs1-3 increased beta4 mRNA, protein, and cell surface expression. The active E2Fs were unable to stimulate invasion in cells that expressed a beta4 short hairpin RNA. This effect of the active E2Fs on beta4 expression does not seem to result from E2F-mediated beta4 transcription because the beta4 promoter lacks known E2F binding motifs. In summary, the data reported here indicate a novel mechanism by which H-Ras can promote the invasion of breast carcinoma cells. This mechanism links active H-Ras, transcriptionally active E2F, and the alpha6beta4 integrin in a common pathway that culminates in enhanced alpha6beta4-dependent invasion