Activation of the transcription factor, AHR, in normal human epidermal keratinocytes increased AHR binding in the gene regions of the glucose transporter, SLC2A1, and the glycolytic enzyme, ENO1. This increased chromatin binding corresponded with AHR-dependent decreases in levels of SLC2A1 and ENO1 mRNA, protein, and activities. Studies of the ENO1 promoter showed activation of the AHR decreases the transcription of ENO1. Glycolysis was lowered by activation of the AHR as measured by decreases in glucose uptake and the production of pyruvate and lactate. Levels of ATP were also decreased. Downregulation of glucose metabolism, either by activation of the AHR, inhibition of glycolysis, inhibition of glucose transport, or inhibition of enolase, increased SIRT1 protein levels in normal human epidermal keratinocytes and the immortalized keratinocyte cell line, N/TERT-1. This increase in SIRT1 was abrogated by the addition of exogenous pyruvate. Moreover, keratinocyte differentiation in response to downregulation of glycolysis, either by activation of the AHR, inhibition of glucose transport, or inhibition of enolase, was dependent on SIRT1. These results indicate that regulation of glycolysis controls keratinocyte differentiation, and that activation of the AHR, by lowering the expression of SLC2A1 and ENO1, can determine this fate.
Cytochrome P4501B1 (CYP1B1) is elevated in breast cancer. Studies indicate a relationship between CYP1B1 and aggressive cancer phenotypes. Here, we report on in vitro studies in triple-negative breast cancer cell lines, where knockdown (KD) of CYP1B1 was used to determine the influence of its expression on invasive cell phenotypes. CYP1B1 KD in MDA-MB-231 cells resulted in the loss of mesenchymal morphology, altered expression of epithelial–mesenchymal genes, and increased claudin (CLDN) RNA and protein. CYP1B1 KD cells had increased cell-to-cell contact and paracellular barrier function, a reduced rate of cell proliferation, abrogation of migratory and invasive activity, and diminished spheroid formation. Analysis of clinical breast cancer tumor samples revealed an association between tumors exhibiting higher CYP1B1 RNA levels and diminished overall and disease-free survival. Tumor expression of CYP1B1 was inversely associated with CLDN7 expression, and CYP1B1HI/CLDN7LOW identified patients with lower median survival. Cells with CYP1B1 KD had an enhanced chemosensitivity to paclitaxel, 5-fluorouracil, and cisplatin. Our findings that CYP1B1 KD can increase chemosensitivity points to therapeutic targeting of this enzyme. CYP1B1 inhibitors in combination with chemotherapeutic drugs may provide a novel targeted and effective approach to adjuvant or neoadjuvant therapy against certain forms of highly metastatic breast cancer.
To determine the cutaneous effects of in utero and lactational exposure to the AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), pregnant C57BL/6J mice were exposed by gavage to a vehicle or 5 μg TCDD/kg body weight at embryonic day 12 and epidermal barrier formation and function were studied in their offspring from postnatal day 1 (P1) through adulthood. TCDD-exposed pups were born with acanthosis. This effect was AHR-dependent and subsided by P6 with no evidence of subsequent inflammatory dermatitis. The challenge of adult mice with MC903 showed similar inflammatory responses in control and treated animals, indicating no long-term immunosuppression to this chemical. Chloracne-like sebaceous gland hypoplasia and cyst formation were observed in TCDD-exposed P21 mice, with concomitant microbiome dysbiosis. These effects were reversed by P35. CYP1A1 and CYP1B1 expression in the skin was increased in the exposed mice until P21, then declined. Both CYP proteins co-localized with LRIG1-expressing progenitor cells at the infundibulum. CYP1B1 protein also co-localized with a second stem cell niche in the isthmus. These results indicate that this exposure to TCDD causes a chloracne-like effect without inflammation. Transient activation of the AhR, due to the shorter half-life of TCDD in mice, likely contributes to the reversibility of these effects.
It is suggested that kallikrein-related peptidases (KLKs) are key players in corneocyte desquamation and this process is regulated by lympho-epithelial Kazal-type inhibitor (LEKTI). However, it is unclear how these proteases are activated and how activated KLKs are released from LEKTI in the upper cornified layer. Previously we reported cloning of a new PRSS3 gene product, keratinocyte-specific mesotrypsin from the cDNA library. It has been reported that mesotrypsin is insusceptible to intrinsic inhibitors. Moreover, we found that mesotrypsin effectively activated pro-KLK5 and pro-KLK7. Although LEKTI strongly inhibited KLKs, mesotrypsin was not suppressed by LEKTI but rather degraded them. Surprisingly, we isolated a novel inhibitor and identified it as serpinB12 (Another name is YUKOPIN). Thin skin, such as the eyelid, showed strong mesotrypsin staining, while serpinB12 staining was barely detectable. In contrast, footpad skin, which has a thick cornified layer, showed strong serpinB12 staining at the granular layer. We constructed skin equivalent models, in which serpinB12 was over-expressed or knocked-down. Cornified layers were increased and the epidermis became thicker in serpinB12 over-expression. When serpinB12 was downregulated, the epidermal structure became very thin. Furthermore, we found that excessive over-expression of serpinB12 induced parakeratosis. Immunohistochemical study showed that serpinB12 was expressed in parakeratic area of diseased skin, such as psoriasis and atopic dermatitis. Our findings provided a new insight into the desquamation mechanisms, in which mesotrypsin and serpinB12 play critical roles. Moreover, our results suggest that serpinB12 would be involved in controlling the thickness of stratum corneum through mesotrypsin regulation, and excessive serpinB12 expression might be the cause of parakeratotic disease condition.
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