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The suppressor of cytokine signaling (SOCS) family of proteins are intracellular mediators of cytokine signaling. These proteins are induced rapidly by cytokine stimulation and act in a classic negative-feedback loop to attenuate the cellular response to the cytokine signal. In this study, we present the cloning and initial characterization of the Xenopus SOCS3 gene. We show that xSOCS3 is rapidly induced in response to epithelial wounding in the tadpole. The induction of xSOCS3 in response to trauma is transient with maximal expression being reached 1 hr after the injury and diminishing after that. Unlike other genes known to be responsive to wound-induced activation of the mitogen-activated protein (MAP) kinase pathway, such as Egr1, SOCS3 expression in response to trauma is unaffected by blockade of the MAP kinase pathway by chemical inhibitors.
Figure 2. Developmental expression of suppressor of cytokine signaling-3 (SOCS3). Total cellular RNA from staged embryos was used as to produce first-strand cDNA template for the amplification reaction. Ornithine decarboxylase (ODC) was used as control for RNA recovery. Numbers represent the Nieuwkoop and Faber stage of development. âno RNAâ is a negative control that lacked template.Download figure to PowerPoint
Figure 3. Suppresor of cytokine signaling-3 (SOCS3) expression is rapidly induced after epithelial wounding. A: Embryos were subjected to superficial epithelial wounds, allowed to heal for the stated times (0–8 hr) and then subjected to in situ hybridization using a SOCS3 antisense riboprobe. SOCS3 is first evident at 30 min after trauma and is maximal at 1 hr. The wound-induced expression of SOCS3 is transient and dramatically down-regulated 2 hr after trauma and is undetectable by 8 hr after trauma. B: SOCS3 is rapidly induced after trauma at early stages of embryonic development. Embryos at the gastrula stage (stage 11) were wounded and allowed to heal for 1 hr before fixation and in situ hybridization for SOCS3 expression. C: SOCS3 expression is induced in the epidermis and underlying tissues after trauma. Cross-section (8 μm) of a stage 28 embryo after wounding, healing for 1 hr, and then analyzed for SOCS3 expression by in situ hybridization. SOCS3 expression (blue staining) in evident in the epidermis and the underlying tissues. Arrow indicates the site of wounding, and the parallel dotted lines indicate the depth of the epidermal layer. Original magnification, ×200. N, notochord; S, somite; fg, foregut; e, epidermis. Download figure to PowerPoint
Figure 4. A: Extracellular signal-regulated kinase (ERK) is activated rapidly after epithelial trauma. Embryos were wounded and allowed to heal for 2 or 10 min before fixation and immunohistochemistry for activated (bis-phosphorylated) ERK. ERK activation is evident within 2 min after trauma and is extensive after 10 min. B: Trauma-induced ERK activation is blocked by the mitogen-activated protein/ERK kinase (MEK1) inhibitor PD98059. Embryos were pretreated for 2 hr in 0.1× MMR containing either PD98059 or AG490 or SB202190 (each at 50 μM final concentration; see Table 1) before trauma. After 10 min, the embryos were fixed and immunostained for activated ERK. Inhibition of MEK1 by PD98059 resulted in a block in ERK activation after injury. Inhibition of JAK2 (AG490) or p38 (SB202190) did not affect the activation of ERK after trauma. C: Wound-induced SOCS3 expression is not inhibited by blocking MEK1 activity. Embryos were pretreated for 2 hr in 0.1× MMR containing either PD98059 or AG490 or SB202190 (each at 50 μM final concentration; see Table 1) before trauma. After 1 hr, the embryos were fixed and SOCS3 expression was assayed by in situ hybridization. Blocking MEK1 activity had no affect on the induction of SOCS3 expression at the site of epithelial wounding.Download figure to PowerPoint
