Serine-arginine-rich (SR) or SR-like splicing factors interact with exon junction complex proteins during pre-mRNA processing to promote mRNA packaging into mature messenger ribonucleoproteins (mRNPs) and to dictate mRNA stability, nuclear export, and translation. The SR protein family is complex, and while many classical SR proteins have well-defined mRNA processing functions, those of other SR-like proteins is unclear. Here, we show that depletion of the homologous non-classical serine-arginine-rich (SR) splicing factors Bcl2-associated transcription factor (Btf or BCLAF) and thyroid hormone receptor-associated protein of 150 kDa (TRAP150) causes mitotic defects. We hypothesized that the depletion of these SR-like factors affects mitosis indirectly through an altered expression of mitotic checkpoint regulator transcripts. We observed an altered abundance of transcripts that encode mitotic regulators and mitotic chromosome misalignment defects following Btf and/or TRAP150 depletion. We propose that, in addition to their previously reported roles in maintaining mRNA distribution, Btf and TRAP150 control the abundance of transcripts encoding mitotic regulators, thereby affecting mitotic progression in human cells.
Objective: People with combined ischemic and diabetic wounds of the lower extremities have the highest risk for limb loss, especially for those without surgical revascularization options. We have demonstrated that Poly-ADP-Ribose polymerase (PARP-1) is hyperactivated in hyperglycemic/hypoxic cells and in ischemic/diabetic murine wounds. This study elucidates the molecular mechanisms of PARP-1 mediated impairment of angiogenesis in diabetic/ischemic wounds. Methods: A model of dorsal bipedicle flap-ischemic wounds on diabetic mice was used. The wounds were treated topically with nanoparticle-encapsulated siPARP-1 or vehicle. Wound closure rate and perfusion was analyzed using digital photography and Laser Doppler scanning, respectively. Angiogenetic markers in the tissues were measured by immunohistochemistry. In-vitro endothelial tube formation assay was performed using HUVECs cultured under hyperglycemic and hypoxic conditions. Results: Wounds treated with topical siPARP-1 significantly accelerated wound healing compared to vehicle (from 25% ± 5% to 40%± 8% ( n =7, p < .05) by day 6 and from 50% ± 15% to 75%± 3% ( n =7, p < .05) by day 12, and also exhibited improved tissue perfusion (50%± 5% increase in perfusion units over control on day 6, n =47 p <0.05). Improved capillary density was also observed in the siPARP-1 treated wounds detected by immunohistochemistry for SMA (250%±35% increase in mean fluorescence intensity over control on day 12, n =4, p <0.05) and CD31 (125% ± 15% increase in mean fluorescence intensity over control on day 12, n =4, p <0.05). In-vitro angiogenesis assay showed that PARP-1-silencing significantly enhanced endothelial tube formation of hyperglycemic/hypoxic HUVECs (15± 4 complete polygons as compared to 0 in untreated, n =4, p <0.05). Human angiogenesis PCR-array analysis of pro-angiogenic factors revealed that PARP-1 silencing upregulated FOSL1 transcription by 5-fold ( n =4, p <0.05). Interestingly, co-silencing of FOSL1 in PARP-1 silenced HUVECs resulted in loss of endothelial tube formation. Conclusions: PARP-1 silencing is an effective strategy to promote ischemic-diabetic wound healing. Our data suggest that PARP-1-FOSL1 is a potential novel axis in angiogenesis and PARP-1 could be a promising therapeutic target for improving angiogenesis in these wounds.
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