Received Date: December 10, 2015; Accepted Date: February 12, 2016; Published Date: February 15, 2016
Citation: Dvashi Z, Rosner M, Stein R, Ziv H, Pollack A et al (2016) Aberrant Activity of TAK1 is Associated with Retinal Pathology. J Cytol HistolS5:007. doi:10.4172/2157-7099.S5-007
Copyright: © 2016 Pollack A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Transforming growth factor-β-activated kinase-1 (TAK1) is a mitogen activated protein kinase kinase kinase that is involved in diverse biological roles across species. Functioning downstream of TGF-β, TAK1 mediates the activation of the c-Jun N-terminal kinase (JNK) signaling pathway, serves as the target of pro-inflammatory cytokines, such as TNF-α, mediates NF-κβ activation, and plays a role in Wnt signaling in mesenchymal stem cells. Still, the expression of TAK1 in the retina has not been defined. In our study, pathological and immunohistochemical assessments indicate a link between retinal pathology and TAK1 phosphorylation. We observed similar TAK1 expression both in non-obvious and obvious retinal pathologies. However, the phosphorylated form of TAK1 in the segments of retina with obvious pathology was hardly detected compared to its expression in the segments with non-obvious pathology. This finding indicates, for the first time, a possible involvement of TAK1 in human retinal pathologies. Better understanding the expression pattern of TAK1 may serve as a new therapeutic avenue for retinal pathologies.
Transforming growth factor-β-activated kinase 1 (TAK1); Retinal pigment epithelium (RPE); Phosphorylation
Transforming growth factor-β-activated kinase 1 (TAK1) is a MAP3K (MAP kinase kinase kinase) which activates the p38, JNK-MAPKs (c-Jun N-terminal protein kinase of mitogen-activated protein kinases) and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) pathways following various stimulations [1-3]. TAK1 is activated downstream of several receptors and cascades, such as transforming growth factor-β (TGF-β), Toll-Like receptors, Interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-α), T-cell receptor and B-cell receptor, all are inflammation-associated cytokines and chemokines . Upon binding by their corresponding ligands, these receptors attract TNF receptor associated factors (TRAFs), which activate TAK1 .
TAK1 was originally identified as a MAPK kinase kinase 7 (MKKK7 or MAP3K7) in the TGF-β signaling pathway, which can also be activated by environmental stress. For TAK1 activation, phosphorylation at Thr-187 and Ser-192 residues in the activation loop of TAK1 is essentially required . TAK1 transduces signals to several downstream signaling cascades, including the MAPK kinase (MKK) 4/7-JNK cascade, MKK3/6-p38 MAPK cascade and nuclear factor κB (NF-κB)-inducing kinase-IκB kinase cascade . Recently, it has been shown that TAK1 is a major mediator of TGF-β1-induced type I collagen and fibronectin expression through activation of the MKK3- p38 MAPK and MKK4-JNK signaling cascades, respectively . Furthermore, increased expression and activation of TAK1 enhances p38 phosphorylation and promotes interstitial fibrosis . These data ascribe a crucial role to TAK1 in extracellular matrix production and tissue fibrosis. TAK1 is also implicated in regulation of cell cycle, cell apoptosis, inflammation, and in the Smad signaling pathway [10,11]. Thus, TAK1 may function as an important regulator and mediator of TGF-β1-induced Smad-dependent and Smad-independent signaling pathways.
Acting upstream in these crucial signal-transduction pathways, TAK1 has been the subject of numerous studies, both in vitro and in vivo. However, a comprehensive study of TAK1 function is challenging, since its ablation leads to embryonic lethality [12,13]. Studies using cell-type specific conditional knock-out mice revealed that TAK1 regulates inflammatory and fibrotic responses in skin and others tissues . In addition, an inducible knock-out TAK1 deletion was shown to promote bone marrow and liver failures due to massive apoptotic cell-death of the bone marrow and hepatocytes .
In human pathology, TAK1 is often mentioned in the context of autoimmune diseases. Numerous publications have demonstrated a crucial role for TAK1 in pro-inflammatory signaling, such as those induced by TLRs, NOD2, IL-1β, TNF-α, T-cell and B-cell receptors [16,17]. In addition, previous studies performed in our laboratory have shown that TAK1 expression in human retinal pigment epithelial (RPE) cells was high and altered following oxidative stress. Moreover, TAK1 inhibition led to reduction in cell proliferation, cell-cycle arrest at G0/G1, and increased SA-β-gal expression, all known to be features of cell senescence . Exposure of cells to oxidative stress combined with inhibition of TAK1 activity decreased the expression of pro-apoptotic proteins, such as p53, and promoted cellular senescence . The study hereby aims to further question TAK1 pattern of expression in retinal pathologies by using specimens from enucleated eyes.
The study adhered to the tenets of the declaration of Helsinki and was performed in compliance with the Health Insurance Portability and Accountability Act. Institute: Sheba Medical Center, Permit ID number-SMC-13-0830.
Human retinal specimens were collected from enucleated eyes. The causes of enucleations were either blind painful eyes or intraocular tumors. The state of blind painful eyes resulted from numerous pathologies, including retinal ischemia. In contrast, intraocular tumors are usually isolated retinal findings which affect only well-defined retinal segments, leaving the unaffected retina healthy. Hence, tumor-free retinal segment from these eyes may represent healthy retina.
We defined the retinas taken from blind painful eyes as having obvious pathology, whereas the segments of retina with normal appearance, from eyes with intraocular tumors, were defined as specimens with non-obvious retinal pathology.
To study TAK1 possible involvement and to characterize its expression pattern in human retinal pathologies, we examined TAK1 expression and activity in both obvious and non-obvious retinal pathology specimens.
The specimens from either group were embedded in paraffin, and 4 μm sections were boiled for antigen retrieval (10 mM citrate buffer 20 min), rinsed in PBS, incubated for 1 hour with 0.2% tween and gelatin in PBS, washed with PBS, blocked with 2% normal goat serum in 1% BSA and 0.1% TritonX100 for 30 min and then incubated overnight with primary antibodies diluted in PBS. Antibodies used: rabbit polyclonal phospho-Thr187 TAK1 (Bioss, Woburn, MA, USA); rabbit polyclonal TAK1 (Novus, Littleton, CO, USA); Isotype control rabbit polyclonal. Following primary antibody reaction the slides were washed with PBS, incubated in H2O2 for 10 min, stained with Histofine Simple Stain TM MAX PO (Nichirei, Tokyo, Japan) for 10 min at room temperature (RT). Following additional washing in PBS, the sections were stained using HRP-streptavidin for 10 min at RT, washed with PBS and incubated at RT with diaminobenzidine for 10 min, washed and counterstained with hematoxylin for 2 min at RT, washed in H2O, dehydrated in graded alcohol (70-100%) and finally mounted on coverslips. Slides were photographed by Olympus bx51 microscope and and Cell^D software (Olympus, Modiin, Israel).
TAK1 and phospho-TAK1 expressions were determined according to the intensity of the signal in the different retinal layers. Not detected, + small levels of expression, ++ medium levels of expression, +++ high levels of expression in all layers.
Ten retinal specimens from ten enucleated eyed of ten patients were evaluated, five from blind painful eyes (representing obvious retinal pathology), and five from eyes with intraocular tumors (representing non-obvious retinal pathology). The age of the patients and their ocular clinical diagnosis are listed in Table 1.
|Segment with obvious Retinal pathology|
|Blind, painful eye (clinical diagnosis unknown)||male||87||1|
|Blind Eye, Choroidal hemorrhage following traumatic perforation||female||82||2|
|Blind eye, neovascular glaucoma following CRVO*||male||72||3|
|Blind eye, Infectious choroiditis||male||85||4|
|Blind eye, Glaucoma and CRVO*||male||71||5|
|Segment with non-obvious Retinal pathology|
|Invasive Basal Cell Carcinoma (BCC)||female||82||9|
*CRVO = central retinal vein occlusion
**BCC = Invasive Basal Cell Carcinoma
Table 1: Characteristics of patients' enucleated eyes.
TAK1 expression in human retinal specimens
Several retinal pathologies induce aberrant activation of MAP kinase proteins . However, very little is known about TAK1 involvement and expression in human pathologies. In this study we have characterized TAK1 pattern of expression in non-obvious pathological segments and in obvious pathological segments of the different retina.
TAK1 was found to be highly expressed in the outer nuclear layer, inner nuclear layer, ganglion cells, choroidal endothelial cells and RPE cells of all retinal specimens (Figure 1A and Table 2). In most cells TAK1 expression was localized mainly in the nucleus. This pattern of expression was demonstrated both in segments of non-obvious retinal pathologies and segments from obvious pathologic retina specimens (Figures 1A and 1B).
|Segment with obvious Retinal pathology|
|Phospho-TAK1 expression||TAK1 expression||Clinical diagnosis||Patient|
|Not detected||+++||Blind, painful eye (clinical diagnosis unknown)||1|
|Not detected||+++||Blind eye, Choroidal hemorrhage following traumatic perforation||2|
|+||+++||Blind eye, Neovascular glaucoma following CRVO*||3|
|Not detected||+++||Blind eye, Infectious choroiditis||4|
|Not detected||+++||Blind eye, Glaucoma and CRVO*||5|
|Segment with non-obvious Retinal pathology|
|Phsopho-TAK1||TAK1 expression||Pathology Clinical diagnosis||Patient|
*CRVO = central retinal vein occlusion
** BCC = Invasive Basal Cell Carcinoma
Table 2: TAK1 and phospho-TAK1 expression in retinal specimens.
Figure 1: Aberrant Activity of TAK1 in Human Pathologic Retina. A and B: Retinal specimens, were immunostained with anti-TAK1 antibodies and hematoxylin (TAK1 expression is manifested by brown staining and black arrows), blue- hematoxylin. C and D: Retinal specimens were treated as in A and stained with phospho- Thr 187 TAK antibodies (brown) and hematoxylin (blue), demonstrating aberrant activity of TAK1 in the retina of blind painful eyes. IR-inner retinal layers; INL- inner nuclear layer; OPLouter plexiform layer; ONL- outer nuclear layer; S- photoreceptor segment (Scale bar = 50μm).
In contrast, the expression of activated TAK1, detected by phospho- Thr 187 TAK1 antibodies, was different between the obvious and non-obvious pathological specimens. The presence of activated TAK1 was found in all layers of the normal retina, with highest levels in the inner nuclear cells (Figure 1C). However, in the pathologial retina phospho- TAK1 was hardly detected (Figure 1D).
TAK1 expression in the RPE cells
RPE cells play a key role in regulating the onset and progression of numerous retinal diseases, including age related macular degeneration (AMD) and proliferative vitreoretinopathy . Consequently, we focused on the expression of TAK1 in the RPE cells. As can be seen in Figure 2A, in the healthy RPE cells the levels of TAK1 are moderate and mostly detected in the nucleus. Furthermore, the levels of phsopho-TAK1 are also mostly detected in the nucleus of the RPE cell. Interestingly, while TAK1 expression was obvious and seen in the pathological tissue (Figure 2B and 2C), activated TAK1 was not detected in the RPE cells of these specimens (Figure 2D).
Figure 2: Attenuated TAK1 Activity in RPE Cells with Pathology. Attenuated TAK1 Activity in RPE Cells with Pathology. A and B: RPE cells of a retinal specimen were immunostained with TAK1 antibodies and hematoxylin. (TAK1 is manifested by brown staining and black arrows), blue- hematoxylin (N=5). C and D: Retinal specimens treated as in A and stained with phospho-Thr 187 TAK antibodies (brown) and hematoxylin (blue), demonstrating aberrant activity of TAK1 in the RPE cells of blind painful eyes (Scale bar = 50μm). RPE cells of a retinal specimen were immunostained with TAK1 antibodies and hematoxylin. (TAK1 is manifested by brown staining and black arrows), blue- hematoxylin (N=5). C and D: Retinal specimens treated as in A and stained with phospho-Thr 187 TAK antibodies (brown) and hematoxylin (blue), demonstrating aberrant activity of TAK1 in the RPE cells of blind painful eyes (Scale bar = 50μm).
The level of expression of TAK1 in its non-activated and activated states in each of the samples is shown in Table 2.
This study defines for the first time the expression pattern of TAK1 MAP kinase in segments of human retina. Our data reveals TAK1 expression pattern in all human retina layers and demonstrates aberrant activity of TAK1 in human pathologic retinal segments.
TAK1 is a key player in the processes of inflammation and malignancy, regulating numerous downstream proteins . Previous work has shown that TAK1 is expressed ubiquitously during early development of mice. During mid-gestation, TAK1 expression becomes more restricted, with high levels seen specifically during development of diverse organs and tissues, including the nervous system, testis, kidney, liver, gut, lung and pancreas , suggesting TAK1 may play multiple roles in mouse organ development. Of note, the expression of TAK1 detected in this study was mostly restricted to the cell nucleus. A recent publication indicated that TAK1 is expressed in the murine cochlea and that its expression pattern changes as the cochlea matures . Whereas TAK1 was broadly expressed in both the developing otocyst and periotic mesenchyme in the murine embryo, by adulthood TAK1 labeling was limited to the pillar cells and the supporting cells in direct contact with inner and outer hair cells .
In contrast, very little is known regarding TAK1 expression in human cells. In human pathology, TAK1 is often mentioned in the context of autoimmune disease. . In addition, TAK1 is involved in the activation of rheumatoid arthritis synoviocytes and human articular cartilage .
In the current study, the specimens of obvious pathologic retina were obtained from enucleated blind painful eyes. The causes for blind painful eye in these cases were diverse, including trauma, end stage glaucoma and retinal vein occlusion (as can be seen in Table 1). The specimens with presumed normal retina were obtained from segments of retina from eyes enucleated due to intraocular malignancy or orbital tumors. Segments of retina located away from the tumor were used; these segments are unaffected by the tumor and contain intact retina.
We found that the expression of TAK1 is manifested mainly in the nuclei of the outer nuclear layer, inner nuclear layer, ganglion cells, choroidal endothelial cells and RPE cells. Similar expression patterns were detected in both normal and pathologic retinas. It is well known that TAK1 activation is stimulated by phosphorylation on threonine 187 residues upon inflammatory response, stress or malignancy . Therefore, this study examined the activity of TAK1 utilizing specific antibodies recognizing the phosphorylation on Thr-187 residue. The data presented hereby reveals that while TAK1 is present in both normal and pathologic retinas, its activity in the obvious pathological retina is barely detected in the outer nuclear layer, inner nuclear layer, ganglion cells and choroidal endothelial cells, and is further diminished in the RPE cells.
Our findings regarding TAK1 expression and activity in the human retina place it as a key factor in retinal pathologies. Even though there is diversity in the etiology of the different cases with blind painful eye, the same pattern of expression in the activated form of TAK1 was observed. We suggest that the retinal pathology may attenuate TAK1 activity, thus contributing to the deterioration of the retina; however, we cannot rule out the possibility that aberrant activity of TAK1 contributes to the onset and/or the progression of retinal pathologies. Still, these assumptions need to be further studied to reveal the exact role of TAK1 in retinal pathophysiologies.
In conclusion, this study demonstrates for the first time the pattern of expression of TAK1 in the human retina and characterizes its activity expression in the human pathologic retina. Nevertheless, additional study is required in order to delineate the mechanism by which TAK1 participates in the different retinal pathologic disorders. Elucidating the role of TAK1 in the human retina and other ocular tissues may be used as a potential therapeutic avenue for ocular disorders.
We would like to thank Dr. Monica Huszar and Ayelet Harari from the pathological department in Kaplan Medical Center for all their help in this article.