The Journal of biological chemistry
-
The homeodomain transcription factor Prrxl1/DRG11 has emerged as a crucial molecule in the establishment of the pain circuitry, in particular spinal cord targeting of dorsal root ganglia (DRG) axons and differentiation of nociceptive glutamatergic spinal cord neurons. Despite Prrxl1 importance in the establishment of the DRG-spinal nociceptive circuit, the molecular mechanisms that regulate its expression along development remain largely unknown. Here, we show that Prrxl1 transcription is regulated by three alternative promoters (named P1, P2, and P3), which control the expression of three distinct Prrxl1 5'-UTR variants, named 5'-UTR-A, 5'-UTR-B, and 5'-UTR-C. ⋯ The most conserved promoter (P3) contains a TATA-box and displays in vivo enhancer activity in a pattern that overlaps with the zebrafish Prrxl1 homologue, drgx. Regulatory modules present in this sequence were identified and characterized, including a binding site for Phox2b. Concomitantly, we demonstrate that zebrafish Phox2b is required for the expression of drgx in the facial, glossopharyngeal, and vagal cranial ganglia.
-
Multiple Ca(2+)-dependent processes are involved in capsaicin-induced desensitization of transient receptor potential vanilloid 1 (TRPV1), but desensitization of TRPV1 by heat occurs even in the absence of extracellular Ca(2+), although the mechanisms are unknown. In this study, we tested the hypothesis that capsaicin and heat desensitize TRPV1 through distinct mechanisms involving distinct structural segments of TRPV1. In HEK293 cells that heterologously express TRPV1, we found that heat-induced desensitization was not affected by the inclusion of intracellular ATP or alanine mutation of Lys(155), both of which attenuate capsaicin-induced desensitization, suggesting that heat-induced desensitization occurs through mechanisms distinct from capsaicin-induced desensitization. ⋯ Within the distal CTD, we identified two segments that distinctly regulated capsaicin- and heat-induced desensitization. The results suggest that the activation and desensitization of TRPV1 by capsaicin and heat can be modulated differentially and disproportionally through different regions of TRPV1 CTD. Identifying the domains involved in thermal regulation of TRPV1 may facilitate the development of novel anti-hyperalgesic approaches aimed at attenuating activation and enhancing desensitization of TRPV1 by thermal stimuli.
-
Macrophage activation is a central event in immune responses. Macrophages undergoing classical activation (M1 macrophages) are proinflammatory, whereas alternatively activated macrophages (M2 macrophages) are generally anti-inflammatory. miRNAs play important regulatory roles in inflammatory response. However, the manner in which miRNAs regulate macrophage activation in response to different environmental cues has not been well defined. ⋯ KLF13 knockdown had similar effects on M1 activation as did miR-125a-5p overexpression. In addition, miR-125a-5p regulates phagocytic and bactericidal activities of macrophages. Our data suggest that miR-125a-5p has an important role in suppressing classical activation of macrophages while promoting alternative activation.
-
Phosphatidylinositol 3-kinases (PI3K) participate in numerous signaling pathways, and control distinct biological functions. Studies using pan-PI3K inhibitors suggest roles for PI3K in osteoclasts, but little is known about specific PI3K isoforms in these cells. Our objective was to determine effects of isoform-selective PI3K inhibitors on osteoclasts. ⋯ These data are consistent with a critical role for PI3Kδ in regulating osteoclast cytoskeleton and resorptive activity. In contrast, multiple PI3K isoforms contribute to the control of osteoclast survival. Thus, the PI3Kδ isoform, which is predominantly expressed in cells of hematopoietic origin, is an attractive target for anti-resorptive therapeutics.
-
The Cryptochrome (CRY) proteins are critical components of the mammalian circadian clock and act to rhythmically repress the activity of the transcriptional activators CLOCK and BMAL1 at the heart of the clock mechanism. The CRY proteins are part of a large repressive complex, the components of which are not completely known. Using mass spectroscopy, we identified the catalytic subunit of DNA-dependent protein kinase as a CRY-interacting protein and found that loss or inhibition of this kinase results in circadian rhythms with abnormally long periods. ⋯ Furthermore, we found that phosphorylation of this site increases the stability of the CRY1 protein and prevents FBXL3-mediated degradation. The phosphorylation of this site is robustly rhythmic in mouse liver nuclei, peaking in the middle of the circadian day at a time when CRY1 levels are declining. Therefore, these data suggest a new role for the C-terminal tail of CRY1 in which phosphorylation rhythmically regulates CRY1 stability and contributes to the proper circadian period length.