Microscopic imaging during immunohistochemical analysis shows a significant expression of KLF4 in the central part of the seminiferous tube containing differentiated cells, while in the basal membrane there is no expression. In addition, images from confocal microscopy during the immunocytochemical analysis of two differentiated and undifferentiated populations of SSCs also indicate that KLF4 is a differentiating factor and its increased expression leads to differentiation of SSCs cells and spermatogenesis. While the results of Yamanaka's experiments and many other researchers after him have shown that KLF4 is essential for somatic cell reprogramming and, in intersection with other proteins, regulates ES cells self-renewal and pluripotency (9, 22). Peilin Zhang et al. Also stated that Klf4, the same as Oct4, NANOG, and Sox2, is highly expressed in ES cells, while its expression decreases dramatically during differentiation (23). Studies by Yan Sun et al. On the association of KLF4 with NANOG have also shown that NANOG expression levels are directly controlled by KLF4, which prevents differentiation in ES cells, and on the other hand KLF4 itself is directly activated by Oct4 and Stat3 and the overexpression of these factors increased the amount of Klf4 transcripts .(24) The results obtained from Fluidigm analysis show a significant decrease in KLF4 expression in passage 10 of undifferentiated SSCs in comparison to passage 0, which indicates that KLF4 expression has decreased with increasing self-renewal and proliferation. Similar to the results of our experiments, Dandan Yang et al. has found that during the knockout of KLF4 in the testis of Chlamys farreri, the spermatogenesis process was disrupted and it has even been associated with the change of gender from male to female, proving the crucial role of KLF4 in differentiation and spermatogenesis (25). Kit-Ling Sze et al. Has shown that KLF4 is involved in the activation of some proteins involved in tight junction, so it appears to regulate the movement and translocation of germ cells to cross the blood-testis barrier (26). Deletion of KLF4 has also been shown to impair Sertoli cell morphology during puberty in mice, although it does not impair fertility (27-29). From another perspective, we can point to the oncogenic role of KLF4 in cancer, which has been reported to be required for the maintenance of cancer stem cells (CSCs) in breast ductal carcinoma and prostate cancer. It also sustains and develops tumors in oral squamous carcinoma, and head and neck cancer (30). However, decreased klf4 expression in gastric, lung, and colon cancers has also been reported, and KLF4 expression levels are lower in cancer cells than in adjacent non-cancerous tissues, and its expression is inversely related to tumorigenesis, which supporting the role of KLF4 as a tumor suppressor. A combination of these studies proves that, KLF4 can play a dual role depending on different cellular contexts and types of cancer. This behavior is similar in other factors such as TGF-β (27, 30).
The complex telomerase activity is dependent on TERT, which has RNA-dependent RNA polymerase (RdRP) activity, and its gene expression is required to maintain or reactivate telomerase. As a result, it is one of the momentous factors in regulating mitotic progression and the characteristics of stem cells as well as cancer stem cells (11, 31). To ensure that TERT is silenced in most normal cells but expressed in a timely manner and a proper place in a small group of cells such as SSCs, many factors, indirectly or directly, regulate TERT transcription in collaboration with transcription factors or other regulatory elements in a context-dependent manner; Therefore, the TERT promoter can have multiple and dynamic outputs in response to a variety of signaling paths (14, 32, 33). Recent studies have shown that WNT signaling is associated with TERT due to the cofactor role of TERT for β-catenin (Ctnnb1) (11). The TERT-BRG1 association has been shown to be involved in modulating transcription of Wnt target genes such as b-catenin (34). Wnt signaling is one of the essential growth, development, differentiation, stemness regulatory cascades and plays a critical role in fetal development and maintenance of cellular homeostasis. Hence, its mechanism is highly conserved in terms of evolution. However, its improper regulation is also associated with various inflammatory-related cancers (35). The canonical Wnt pathway is β-catenin-dependent, although it can also be non-β-catenin-dependent, which is called the non-canonical signaling pathway (36). β-catenin is the major nuclear agent in the WNT signaling pathway, and if it is present in the cytoplasm freely and not attached to E-cadherin, it will be destroyed by a complex consisting of Axin, APC, glycogen synthase kinase-3b (GSK3b) and casein kinase 1a (CK1a) that does not allow β-catenin to accumulate (37). Initiation of Wnt signaling leads to the accumulation and nuclear transport of the stable β-catenin that could affect transcriptional activators or the expression of transcription factors (37, 38). The Wnt/β-catenin signaling pathway has been shown to induce the differentiation of pluripotent embryonic stem cells into mesoderm and endoderm progenitor cells, which during this process, β-catenin is increased nuclear and cytoplasmic that leads to the activation of transcription of proteins such as cyclin D1 and c-Myc. These two proteins play a crucial role in controlling the cell cycle's G1 to S phase transition. And since cell proliferation is directly related to stem cell differentiation, the Wnt cascade is active during differentiation, and the Ctnnb1 is considered a differentiating factor (39-41). In this case, according to the results of our experiments, it seems that KLF4 is aligned with Ctnnb1, and both have increased expression during the differentiation process of SSCs. So, under these circumstances, the effect of Wnt signaling on TERT expression and telomerase activity is indirect, in which the c-Myc transcription factor directly controls TERT transcription and is a transcriptional stimulus of TERT expression, which is also regulated by Wnt/β-catenin signaling (38, 42). But extensive studies on this cascade have shown that the Wnt pathway connects to TERT in a slightly different way. Nusse Roel et al. (43) has identified the Wnt pathway as a main cascade in maintaining and regulating self-renewal and pluripotency in embryonic stem cells. In addition, Peter Wend et al. had endorsed the Wnt pathway support in the formation and maintenance of cancer stem cells (44). On the other hand, according to data collected by Hoffmeyer et al., by the β-catenin deficiency in ESCs and adult stem cells, TERT expression decreases dramatically; therefore, Stem cells have a higher expression of TERT because they have higher β-catenin expression. Their findings demonstrate the importance of β-catenin expression and the activation of the Wnt pathway to maintain stemness. The authors also showed that β-catenin binds to the TERT promoter so that in vivo β-catenin expression directly increases TERT transcription. Therefore, c-Myc no longer plays a role in this regulatory process. According to these studies, Ctnnb1 expression levels directly affect TERT expression and telomerase activity. On the other hand, it has been suggested that TERT expression also affects Ctnnb1 expression reciprocally, and klf4 also cooperates in this regulatory loop (45, 46). According to the evidence obtained in our experiments, the expression of KLF4 protein increases during differentiation, so if we consider that the Wnt pathway is active in stem cells, then during the differentiation, the expression of genes involved in this signaling must be decreased. So, it seems that as KLF4 increases, the Wnt signaling pathway shuts down. Now the question arises: How does KLF4 affect the Wnt signaling pathway, and what is the KLF4 regulatory role?
Guang-Jun Zhang et al. has found that MiR-92a activates Wnt signaling during colorectal cancer. MiR-92a does this stimulation directly by downregulating KLF4 and promoting stem cell-like properties in CSCs. This finding also confirms the anti-cancer role of KLF4. And it appears that KLF4 suppressed the Wnt pathway by inhibiting the β-catenin transactivating domain in CRC cells (47). Many studies have also shown that KLF4 can bind to the TERT promoter and control TERT expression levels in cooperation with β-catenin (38, 45, 46). Thus, the findings generally suggest that the regulation of TERT expression requires two central transcriptional regulators, KLF4 and β-catenin, to ensure that there is not too much that may allow cancer cells to grow or lead to a lack of differentiation of SSCs, and not too little, which may lead to the destruction of stem cells in the body.