*3.3.1 KLF4 regulates FSP-1 in fibrocyte generation from MDSCs*

FSP-1, also known as S100A4, is widely accepted as a fibroblast-specific marker [77, 78]. Given the fact that FSP-1 is expressed in more than 90% of monocytes of the host immune system [79] and that it has a "specific" expression in fibroblasts, it is challenging to reconcile the function of FSP-1 at the cellular level between these two very different cell types. On the other hand, fibrocytes are bone marrowderived progenitor cells that can differentiate into myofibroblasts and promote cutaneous would healing and cancer development [20, 51, 80, 81]. Therefore, fibrocytes are very good candidates for carrying the expression/function of FSP-1 from the host immune cells such as MDSCs to fibroblasts.

It has been reported that fibrocytes can be generated from bone marrow-derived cells such as MDSCs [82]. We postulated that KLF4 controls MDSC-mediated generation of fibrocytes. To test this hypothesis and to examine the underlying mechanisms, we isolated spleen cells from KLF4 inducible knockout Rosa26CreER/ KLF4(flox) mice and examined fibrocyte differentiation using an *ex vivo* assay with murine IL-13 and M-CSF [83]. We found that the application of IL-13 and M-CSF resulted in 58 ± 7 fibrocytes per 1 × 105 cells (**Figure 1A**) in the control group. However, the same treatment decreased the number of fibrocytes to 5 ± 2 cells per 1 × 105 splenocytes when KLF4 was knocked out by induction of 5 μM 4-OH tamoxifen (**Figure 1B**). Furthermore, we examined KLF4 and FSP-1 expression in the process of fibrocyte generation by quantitative RT-PCR analysis. As shown in **Figure 1C**, both KLF4 and FSP-1 mRNA levels were significantly elevated after the application of IL-13 and M-CSF, which was consistent with *ex vivo* generation of fibrocytes. The induction of KLF4 deficiency by 4-OH tamoxifen correlates with a significant decrease in FSP-1 expression, suggesting a KLF4-mediated regulation of FSP-1 in the process. Since splenocytes are a mixed group of cells, we proceeded to examine KLF4 and FSP-1 expression in different subsets of MDSCs from the wild type mouse splenic tissues (**Figure 1D**). Highest levels of KLF4, FSP-1, and CCR2 expression were found in the CD11b+ Ly6GInt subpopulation of MDSCs (P2 in **Figure 1D** and **E**), known as M-MDSCs [84, 85]. Note that these M-MDSCs had the highest potential for fibrocyte generation (**Figure 1F**), thus supporting the observation that KLF4 deficiency led to significant decrease in FSP-1 expression and fibrocyte generation (**Figure 1A–C**) in the MDSC pool. To test whether KLF4 directly regulates FSP-1 gene expression, we first using two different KLF4 antibodies to perform a chromatin

#### **Figure 1.**

*KLF4 regulates FSP-1 gene expression in fibrocyte generation. (A) Representative photographs of morphological fibrocyte generation from splenocytes in the absence and presence are indicated by red arrows. KLF4 deficiency was induced by 4-OH tamoxifen (TAM). (B) Quantification of the data from (A). (C) Relative levels of KLF4 and FSP-1 mRNA in fibrocyte generation as assessed by qRT-PCR. (D) Different MDSC subsets in mouse splenocytes measured by flow cytometry. (E) Relative levels of KLF4, FSP-1 and CCR2 mRNA in different MDSC subsets by qRT-PCR. (F) Potential of fibrocyte generation from MDSC subsets in mouse spleen. (G) Left—binding of KLF4 to the FSP-1 promoter as assessed by chromatin immunoprecipitation assay using two KLF4 antibodies (KLF4-1 and KLF4-2). IgG was used as a negative control. Right—the effect of KLF4 overexpression on FSP-1 promoter activities, as examined by transient transfection and dual luciferase assays, \*P < 0.05, \*\*P < 0.01.*

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*KLF4-Mediated Plasticity of Myeloid-Derived Suppressor Cells (MDSCs)*

direct regulation of FSP-1 by KLF4 at the transcriptional level.

KLF4 is involved in epigenetic control of MDSC subsets or plasticity.

*3.3.3 Is there potential molecular plasticity of KLF4 in cancer and wound healing?*

KLF4 is a transcription factor with multiple functions in different physiological and pathological conditions, notably in cancer development. For example, KLF4 is well known for its tumor suppressive effect on tumor development in the gastrointestinal tract [102]. However, high expression of KLF4 is associated with skin cancer and breast cancer development [56, 103, 104], suggesting a tumor promoting function of KLF4 in these tissues. Recently, a tumor suppressive function of KLF4 was also reported in breast cancer [105]. These contradictory reports suggest context-dependent functions of KLF4 in cancer development [106]. At a molecular level, different KLF4 transcripts were found in testis [107], and alternative splicing of KLF4 has been proposed to explain context-dependent functions of KLF4 [108]. Consistently, an oncogenic KLF4 isoform, named KLF4α, has been found in both pancreatic cancer [109] and breast cancer [110]. In line with these observations, there is dynamic expression of KLF4 isoforms in mouse embryogenesis [111].

immunoprecipitation (CHIP) assay. We found that KLF4 directly bound to the FSP-1 proximal promoter region (**Figure 1G** left). Then we constructed a FSP-1 promoter luciferase reporter containing ~2.3 kb of the FSP-1 promoter region. By transient transfection and dual luciferase assays, we found that KLF4 overexpression resulted in three fold increase of the FSP1 promoter activity (**Figure 1G** right), suggesting a

The studies of epigenetics, heritable changes to gene expression without changes to DNA, are significantly advancing our knowledge of the inflammatory conditions [86]. They include DNA modifications mainly methylation, histone tail modifications, and non-coding RNA-mediated gene regulation. Recent data revealed that epigenetic mechanisms could provide novel strategies for modulating wound

Critical functions of KLF4 have been shown in the generation of induced pluripotent stem cells and in cancer development through epigenetic mechanisms [90, 91]. In addition, there are numerous reports showing that microRNAs regulate KLF4 [92– 94] or KLF4 regulate microRNAs [95, 96] in varied pathological conditions. KLF4 mediated DNA methylation have also been reported in hTert promoter [97] and methylation of KLF4 promoter is associated with urothelial cancer progression and early recurrence [98]. Moreover, the correlation of KLF4 and histone modifications has also been reported. For example, histone methyltransferase KMT2D, a frequently aberrant epigenetic modifier in various cancer, sustains prostate carcinogenesis and metastasis via epigenetically activating KLF4 [99]. From the perspective of MDSCs, epigenetic regulation of their differentiation and function is not completely understood. However, there is evidence to indicate the importance of epigenetic regulation. Shang et al. showed that long non-coding RNA retinal non-coding RNA3 (RNCR3) promotes C/EBP homologous protein (Chop) expression by sponging microRNA 185- 5p during MDSC differentiation [100]. In addition, although histone modifications related to myeloid differentiation have been extensively studied [101], currently there is no clear indication about epigenetic markers that can discriminate specific MDSC subsets. Given the role of KLF4 in epigenetic regulation and the importance of MDSC plasticity in cancer and wound healing, it will be very interesting to examine how

*DOI: http://dx.doi.org/10.5772/intechopen.89151*

*3.3.2 Epigenetic control of MDSC plasticity*

healing [87–89].

immunoprecipitation (CHIP) assay. We found that KLF4 directly bound to the FSP-1 proximal promoter region (**Figure 1G** left). Then we constructed a FSP-1 promoter luciferase reporter containing ~2.3 kb of the FSP-1 promoter region. By transient transfection and dual luciferase assays, we found that KLF4 overexpression resulted in three fold increase of the FSP1 promoter activity (**Figure 1G** right), suggesting a direct regulation of FSP-1 by KLF4 at the transcriptional level.
