The mitogen activated protein kinase (MAPK) family is involved in a variety of processes including cellular growth and differentiation (Seger and Krebs, 1995). Signaling through this family occurs when the cell receives a stimulus that activates a mitogen activated protein kinase kinase kinase (MAPKKK).
Within the MAPK family, there is a trio of subfamilies: extracellular signal regulated kinase (ERK), Mitogen activated protein kinase (p38) and c-Jun N Terminal Kinase (JNK). JNK has garnered much interest since this MAPK is involved in a variety of pathways and activated when the cell is exposed to stress, differentiation or growth signals. JNK by application of SP600125 has been used in many studies.
SP600125 inhibits JNK function by pre venting phosphorylation of c-jun which ultimately disrupts normal cellular activity such as autophagy (Zhang et al., 2008) . SP600125 can competitively bind to TCDD (toxin that interferes with normal cellular activity through activation of AhR) responsive elements and activates expression of AhR target genes including CYP1A (Dvorak et al., 2008). To determine the pharmacological impact of SP600125 on zebra fish development, they incubated embryos in various concentrations of SP600125, and see if these different concentrations resulted in abnormalities.
This is the structure of SP600125. Some say it is because of the structure that makes it such a unique inhibitor.
Zebra Fish Maintenance and Chemical Treatments
To determine the pharmacological impact of SP600125 on zebra fish development, they incubated embryos in various concentrations of SP600125 from 18h postfertilization (hpf) to 48hpf.
Whole mount Immunohistochemistry
They did this in order to stain the small pieces of tissue, without sectioning onto slides first. The reason this is optimal is because it allows the ability to follow the expression of target proteins through the development of the animal. This allows the researchers to see the impact the different concentrations of SP600125.
- Embryos in the groups exposed to SP600125 at 0μ M (the controls) and at 1.25μM exhibited 0% mortality rates while 7% of the embryos receiving the 5μ M treat-ment were dead by 120hpf. In addition, application of SP600125.
- External abnormalities were observed in embryos treated with 1.25μ M of SP600125 and became profound when the concentration increased to 5μM or higher.
- Zebra fish treated with 5μ M exhibited numerous developmental defects such as yolk sac edema, reduced body length pericardium edema, deflated swim bladder, bent tail vertebrae, bent trunk vertebrae, and underdeveloped jaws. Deformation also occurs during eye development.
Different Abnormalities caused by SP600125
Full-body images at 4× magnification of 120 hpf larvae exposed to 0 µm (control) (A-C), 1.25 µm (D-F) and 5 µm SP600125 (G-I) during the 18-48 hpf period of embryonic development. Pericardium edema (a) was the sole external defect observed in 1.25 µm-treated fish (D) while many more defects were observed in the 5 µm-treated fish; (b) yolk sac edema; (c) reduced total length; (d) pericardial sac edema; (e) swim bladder deflation; (f) bent tail vetebrae; (g) bent trunk vertebrae; and (h) underdeveloped jaws
- SP600125 at high concentrations severely affected embryonic growth. Embryos were smaller in size and showed delays in hatching in groups exposed to 5and 1.25μ M SP600125. This is seen in the graphs below.
Exposure to the JNK inhibitor SP600125 during early zebrafish development results in morphological defects
Graphs quantitatively depicting the differences between three groups of 120 hpf larvae exposed to 0 (control), 1.25 and 5 µm SP600125 during the 18-48 hpf period of embryonic development. Comparisons of body length (A), retina diameter (B), lens diameter (C) and ratio of lens diameter to retina diameter (D) were made between the three groups. All graphs display highly statistical differences between the control and 1.25 µm-treated fish against those treated with 5 µm of SP600125. ** P < 0.001.
- These features suggest that application of 5μ M SP600125 may have inhibited neuron migration and axon outgrowth, an outcome that correlates with involvement of JNK signaling in neuron growth and survival(Eminel et al., 2008).
- A second experiment with a shorter exposure period of SP600125 (6h) presented significantly fewer morphological defects.
- Under stressful conditions, inhibition of JNK activation causes cell cycle arrests and increases apoptosis, but under optimal conditions, development is normal since JNK is not required for mouse preimplantation embryonic development.
- SP600125 induced toxic side effects at concentrations equivalent to or lower than its effective dosage as a JNK inhibitor.
Effects of varying time and duration of SP600125 exposure during zebrafish development
Larvae of 120 hpf were compared for morphological differences between the control (A) and larvae treated with 5 µm SP600125 for 6 h (B, C) and 30 h (D).
SP600125 caused developmental abnormalities during zebrafish organogenesis starting at 1.25 µm and the defects were exacerbated with increasing concentrations. The study suggests that SP600125 at 1.25 µm and beyond has devastating consequences for zebrafish development. The authors suggest that zebrafish development toxicity was primarily through disruption of signal transduction and neurogenesis, which resulted in reduction of brain size. The authors also suggest that inhibition of JNK signaling is more devastating at a later time during organogenesis because proper regulation of programmed cell death becomes critical in the later stages of organogenesis and JNK signaling involves both pro-apoptotic and anti-apoptotic actions(Dvorak et al., 2008).
Review/Strengths & Weaknesses:
Although the authors did an outstanding job with their experimental design and execution, and presenting their data they were not able to pin point the mechanism that caused all these abnormalities. They could only muster up broad ways in which the abnormalities could have been caused by. Although the authors touched on how this information affected other aspects of development they did not go in much detail. Also the author could have gone in more detail about the interactions SP600125 has on the other members of the MAPK family such as ERK.
Zhang Y, Wu Y, Cheng Y, Zhao Z, Tashiro S, Onodera S, Ikejima T. 2008. Fas-mediated autophagy requires JNK activation in HeLa cells. Biochem. Biophys. Res. Commun. 377: 1205–1210; doi: 10.1016/j.bbrc.2008.10.151.
Dvorak Z, Vrzal R, Henklova P, Jancova P, Anzenbacherova E, Maurel P, Svecova L, Pavek P, Ehrmann J, Havlik R, Bednar P, Lemr K, Ulrichova J. 2008. JNK inhibitor SP600125 is a partial agonist of human aryl hydrocarbon receptor and induces CYP1A1 and CYP1A2 genes in primary human hepatocytes. Biochem. Pharmacol. 75: 580–588; doi: 10.1016/j.bcp.2007.09.013.
Eminel S, Roemer L, Waetzig V, Herdegen T. 2008. c-Jun N-terminal kinases trigger both degeneration and neurite outgrowth in primary hippocampal and cortical neurons. J. Neurochem. 104: 957–969; doi 10.1111/j.1471-4159.2007.05101.x.