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We investigated the signaling basis for tubule pathology during fibrosis after renal injury. Numerous signaling pathways are activated physiologically to direct tubule regeneration after acute kidney injury (AKI) but several persist pathologically after repair. Among these, transforming growth factor (TGF)-β is particularly important because it controls epithelial differentiation and profibrotic cytokine production. We found that increased TGF-β signaling after AKI is accompanied by PTEN loss from proximal tubules (PT). With time, subpopulations of regenerating PT with persistent loss of PTEN (phosphate and tension homolog) failed to differentiate, became growth arrested, expressed vimentin, displayed profibrotic JNK activation, and produced PDGF-B. These tubules were surrounded by fibrosis. In contrast, PTEN recovery was associated with epithelial differentiation, normal tubule repair, and less fibrosis. This beneficial outcome was promoted by TGF-β antagonism. Tubule-specific induction of TGF-β led to PTEN loss, JNK activation, and fibrosis even without prior AKI. In PT culture, high TGF-β depleted PTEN, inhibited differentiation, and activated JNK. Conversely, TGF-β antagonism increased PTEN, promoted differentiation, and decreased JNK activity. Cre-Lox PTEN deletion suppressed differentiation, induced growth arrest, and activated JNK. The low-PTEN state with JNK signaling and fibrosis was ameliorated by contralateral nephrectomy done 2 wk after unilateral ischemia, suggesting reversibility of the low-PTEN dysfunctional tubule phenotype. Vimentin-expressing tubules with low-PTEN and JNK activation were associated with fibrosis also after tubule-selective AKI, and with human chronic kidney diseases of diverse etiology. By preventing tubule differentiation, the low-PTEN state may provide a platform for signals initiated physiologically to persist pathologically and cause fibrosis after injury.

TMEPAI is a transforming growth factor-beta (TGF-beta)-induced transmembrane protein that is overexpressed in several cancers. How TMEPAI expression relates to malignancy is unknown. Here, we report high expression of TMEPAI in estrogen receptor/progesterone receptor-negative and human epidermal growth factor receptor-2-negative breast cancer cell lines and primary breast cancers that was further increased by TGF-beta treatment. Basal and TGF-beta-induced expression of TMEPAI were inhibited by the TGF-beta receptor antagonist SB431542 and overexpression of Smad7 or a dominant-negative mutant of Alk-5. TMEPAI knockdown attenuated TGF-beta-induced growth and motility in breast cancer cells, suggesting a role for TMEPAI in growth promotion and invasiveness. Further, TMEPAI knockdown decreased breast tumor mass in a mouse xenograft model in a manner associated with increased expression of phosphatase and tensin homologue (PTEN) and diminished phosphorylation of Akt. Consistent with the effects through the phosphatidylinositol 3-kinase pathway, tumors with TMEPAI knockdown exhibited elevated levels of the cell cycle inhibitor p27kip1 and attenuated levels of DNA replication and expression of hypoxia-inducible fator 1alpha and vascular endothelial growth factor. Together, these results suggest that TMEPAI functions in breast cancer as a molecular switch that converts TGF-beta from a tumor suppressor to a tumor promoter.

Bone remodeling depends upon proper osteoblast and osteoclast function. Bone morphogenetic protein-2 (BMP-2) stimulates differentiation of osteoblasts from pluripotent precursors. Osteoclast formation depends on the concerted action of osteoblast-derived receptor activator of NF-kappaB ligand and colony-stimulating factor-1 (CSF-1). BMP-2 stimulates receptor activator of NF-kappaB ligand expression. However, the effect of BMP-2 on CSF-1 expression has not been studied. We investigated the role of BMP-2 in CSF-1 expression in osteogenic C2C12 cells. Incubation of C2C12 cells with BMP-2 supported osteoclastogenesis of spleen cells with a concomitant increase in expression of CSF-1 mRNA and protein. To determine the mechanism, we identified a BMP-responsive element between -627 bp and -509 bp in the CSF-1 promoter. DNase I footprint analysis revealed the presence of consensus Smad binding motif in this region. Electrophoretic mobility shift assay showed BMP-2-stimulated binding of proteins to this motif. Mutation of core sequence as well as its 5'- and 3'-flanking sequences abolished the DNA-protein interaction resulting in inhibition of CSF-1 transcription. Supershift analysis detects the presence of Smads 1, 5, and 4 and the transcriptional coactivator CREB-binding protein in the BMP-responsive element-protein complex. In addition, Smads 1 and 5 alone or in combination with Smad 4 increased CSF-1 transcription. Furthermore, CREB-binding protein markedly increased transcription of CSF-1. These data represent the first evidence that BMP-2 increases the osteoclastogenic CSF-1 expression by a transcriptional mechanism using the canonical Smad pathway and provide a mechanism for BMP-2-induced osteoclast differentiation.