Loss of beta1-integrin enhances TGF-beta1-induced collagen expression in epithelial cells via increased alphavbeta3-integrin and Rac1 activity.

Transforming growth factor ß (TGF-ß) promotes tissue fibrosis via the receptor-specific Smad pathway and non-canonical pathways. We recently reported that TGF-ß1-stimulated collagen expression by cultured kidney cells requires integrin-dependent activation of focal adhesion kinase (FAK) and consequent ERK MAP kinase activity leading to Smad3 linker region phosphorylation. Here, we defined a role for αvß3-integrin in this non-canonical pathway. A human kidney tubular cell line in which ß1-integrin was knocked down (ß1-k/d) demonstrated enhanced type I collagen mRNA expression and promoter activity. A second shRNA to either αv-integrin or ß3-integrin, but not to another αv-binding partner, ß6-integrin, abrogated the enhanced COL1A2 promoter activity in ß1-k/d cells. Although αvß3-integrin surface expression levels were not different, αvß3-integrins colocalized with sites of focal adhesion significantly more in ß1-k/d cells, and activated αvß3-integrin was detected only in ß1-k/d cells. Further, the collagen response was decreased by a function-blocking antibody or a peptide inhibitor of αvß3-integrin. In cells lacking αvß3-integrin, the responses were attenuated, whereas the response was enhanced in αvß3-overexpressing cells. Rac1 and ERK, previously defined mediators for this non-canonical pathway, showed increased activities in ß1-k/d cells. Finally, inhibition of αvß3-integrin decreased Rac1 activity and COL1A2 promoter activity in ß1-k/d cells. Together, our results indicate that decreasing ß1 chain causes αvß3-integrin to become functionally dominant and promotes renal cell fibrogenesis via Rac1-mediated ERK activity.

Specific Delivery of Oligonucleotides to the Cell Nucleus via Gentle Compression and Attachment of Polythymidine.

Nonviral delivery of nucleic acids to the cell nucleus typically requires chemical methods that do not guarantee specific delivery (e.g., transfection agent) or physical methods that may require extensive fabrication (e.g., microfluidics) or an elevated pressure (e.g., 105 Pa for microneedles). We report a method of delivering oligonucleotides to the nucleus with high specificity (relative to the cytosol) by synergistically combining chemical and physical approaches. Particularly, we demonstrate that DNA oligonucleotides appended with a polythymidine [poly(T)] segment (chemical) profusely accumulate inside the nucleus when the cells are under gentle compression imposed by the weight of a single glass coverslip (physical; ∼2.2 Pa). Our "compression-cum-poly(T)" delivery method is simple, can be generalizable to three "hard-to-transfect" cell types, and does not induce significant levels of cytotoxicity or long-term oxidative stress to the treated cells when provided the use of suitable compression times and oligonucleotide concentrations. In bEnd.3 endothelial cells, compression-aided intranuclear delivery of poly(T) is primarily mediated by importin ß and nucleoporin 62. Our method significantly enhances the intranuclear delivery of antisense oligonucleotides to bEnd.3 endothelioma cells and the inhibition of two target genes, including a reporter gene encoding the enhanced green fluorescent protein and an intranuclear lncRNA oncogene (metastasis-associated lung adenocarcinoma transcript 1), when compared with delivery without gentle compression or poly(T) attachment. Our data underscore the critical roles of pressure and nucleotide sequence on the intranuclear delivery of nucleic acids.