INTRODUCTION

The regulation of gene expression in response to intrinsic and extrinsic cues is a fundamental cellular process in the growth and development of organisms. Critical to the control of gene expression are transcription factors that bind to specific DNA sequences and subsequently modulate gene transcription 1. A significant number of transcription factors use a conserved zinc finger domain to bind their target DNAs. In fact, the human genome encompasses over 700 genes that contain a particular C2H2-type of zinc finger, which employs two cysteine and two histidine amino acid residues to coordinate the single zinc atom in the finger-like structure 2. A further subgroup of the C2H2-zinc finger proteins exhibits homology to the Drosophila melanogaster segmentation gene product, Krüppel 3. Members of this subgroup are termed Krüppel-like factors (KLFs), and many KLFs exhibit tissue-selective expression and wide-ranging regulatory functions 4. This review will focus on two members of the KLF family of transcription factors, Krüppel-like factor 4 (KLF4) and Krüppel-like factor 5 (KLF5), which are enriched in epithelial tissues but demonstrate contrasting biological activities.

Identification and Initial Characterization of KLF4 and KLF5

A full-length mouse cDNA clone encoding KLF4 (also called gut-enriched Krüppel-like factor or GKLF) was initially isolated from a NIH3T3 cDNA library by reduced-stringency screening with a DNA probe containing the zinc finger region of an immediate early gene product, Zif268 or Egr1 5. Mouse KLF4 contains 483 amino acids, has a predicted molecular weight of 53 kD, and is 90% identical to human KLF4. The carboxyl terminus of KLF4 contains three C2H2-zinc fingers that are most closely related to another member of the family, KLF2 4. KLF4 is a nuclear protein whose cellular address depends on two nuclear localization signals 6. A survey of the tissue distribution in adult mice revealed that KLF4 is highly expressed in terminally differentiated, post-mitotic epithelial cells of the intestinal tract 5, a finding consistent with the anti-prolife-rative effect of KLF4 (see below).

KLF5 (also called intestinal-enriched Krüppel-like factor or IKLF) was initially isolated based on close homology to KLF2 7. The coding region of mouse KLF5 contains 446 amino acids and is 88% identical to the human sequence, previously identified as basic transcription element binding protein 2 or BTEB2 8. Like KLF4, KLF5 is expressed at a relatively high level in the intestinal tract, its expression is concentrated at the base of the crypt epithelium where active cell division occurs 7.

Expression of both KLF4 and KLF5 genes are developmentally regulated, with a higher level of expression occurring toward the later stage of fetal development 9, 10. Depending on the tissues, both overlapping and mutually exclusive patterns of expression have been observed for the two genes. The respective expression of KLF4 and KLF5 in differentiated, post-mitotic villus and proliferative crypt epithelial cells of the adult intestinal tract also occurs during embryonic development 10. The cellular distribution of KLF4 and KLF5 transcripts in the epidermis of the skin in the late stage embryo mirrors that of intestinal epithelium, wherein KLF4 is more highly expressed in terminally differentiated, quiescent suprabasal layer and KLF5 in the proliferative basal layer of the epidermis 10.

KLF4 and KLF5 bind to similar DNA sequences but exert opposing transcriptional regulatory activities

Like many other zinc finger-containing transcription factors, KLF4 and KLF5 bind to cis-DNA elements that are GC-rich. A consensus DNA binding sequence was empirically determined for KLF4 11. This sequence is present in many gene promoters and includes the CACCC element and the basic transcription element (BTE). Indeed, KLF4 was subsequently shown to inhibit the promoter of the cytochrome P-450IA1 (CYP1A1) gene in a BTE-dependent manner 12. In contrast, BTEB2, the human homolog of KLF5, activates transcription through the BTE element 8. KLF4 and 5 have also been shown to antagonize each other in controlling expression of other genes, despite the two sharing very similar, if not identical, cis-DNA sequences 13. An example is the gene encoding KLF4 itself. Here KLF4 is an activator of the KLF4 promoter, while KLF5 is an inhibitor, even though both proteins interact with the same cis-element 13. Moreover, KLF5 abrogates the activating effect of KLF4 on the KLF4 promoter and KLF4 abrogates the inhibitory effect of KLF5 on the same promoter. A potential reason for this competing effect is due to physical competition of the two proteins in binding to a cognate sequence in the KLF4 promoter 13. A similar competing effect between KLF4 and KLF5 in the transcription of several other genes has also been described, including those encoding smooth muscle α-actin 14 and laminin-1 15.

KLF4 and 5 exhibit contrasting effects on cellular proliferation

The specificity of KLF4 expression for terminally differentiated, post-mitotic intestinal epithelial cells, rather than proliferating crypt cells, prompted Shields et al to examine the behavior of KLF4 in cultured cell systems 5. In vitro, the level of KLF4 mRNA correlates with the proliferative state of cells in a manner similar to that seen in vivo; serum-deprived quiescent NIN3T3 cells contain a significant amount of KLF4 mRNA, while actively proliferating cells express little, if any KLF4. When serum-deprived cells are stimulated into proliferation by the addition of fresh serum, the level of KLF4 mRNA decreases. Conversely, KLF4 mRNA levels are increased as proliferating NIH3T3 cells become growth-arrested by either serum starvation or contact inhibition. Finally, forced expression of KLF4 by transfection of proliferating cells results in an inhibition of DNA synthesis 5. From these observations, it was concluded that KLF4 is a growth arrest-associated gene.

A telling mechanism by which KLF4 inhibits DNA synthesis came from studies that examined the response of KLF4 expression to DNA damage-induced growth arrest. DNA damage caused by methyl methanesulfonate (MMS) 16 or γ irradiation 17 leads to the induction of KLF4 expression in a p53-dependent manner. The increase in KLF4 mRNA level parallels the increase in the level of p21WAF1/Cip1 mRNA, a major cyclin-dependent kinase inhibitor 16. Importantly, KLF4 was shown to transactivate the p21WAF1/Cip1 promoter by binding to a specific Sp1-like cis-element in the proximal p21WAF1/Cip1 promoter. This same element is also needed for p53 to activate p21WAF1/Cip1 transcription, although p53 does not directly bind to it. Instead, p53 and KLF4 physically interact with each other, thus allowing p53 to gain access to the p21WAF1/Cip1 promoter and activate p21WAF1/Cip1 transcription 16. A consequence of the p53-dependent activation of p21WAF1/Cip1 expression following DNA damage is an arrest in the cell cycle at both the G1/S and G2/M transition points. KLF4 was shown to be necessary and sufficient in mediating the checkpoint function of p53 at both of these transition points 17, 18. KLF4 accomplishes this task both through its transcriptional activation of p21WAF1/Cip1 and through direct suppression of cyclin D1 19 and cyclin B1 expression 18, which are required for the G1/S and G2/M transitions, respectively. Expression profiling of KLF4 using cDNA microarray analysis confirmed that KLF4 activates transcription of many additional genes encoding inhibitors of the cell cycle and suppresses those encoding promoters of the cell cycle 20. Recent studies from our laboratory indicate that KLF4 is also involved in preventing centrosome amplification after γ irradiation 21 and possibly in preventing aneuploidy after spindle damage (Dalton and Yang, unpublished observations). Taken together, these studies point to a wide-ranging and crucial effect of KLF4 in maintaining the integrity of the cell cycle. The essential role of KLF4 in mediating the DNA damage response of the cell cycle is summarized in Fig. 1.

Figure 1
figure 1

A model by which KLF4 mediates the cell cycle checkpoint functions of p53 in response to DNA damage. The tumor suppressor p53 is activated in response to DNA damage, which in turn activates KLF4. KLF4 then exerts a multitude of effects on expression of downstream genes by activating (→) or inhibiting their expression (). References in support of the specific pathways are as follows: p53 → KLF4 → p21 16; KLF4 cyclin D/Cdk4 19; KLF4 cyclin B/Cdc2 18; KLF4 cyclin E/Cdk2 21; KLF4 STK15 20; KLF4 → 14-3-3 σ ; KLF4 BUB1/MAD2 20.

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The observation that KLF4 exerts an important checkpoint function suggests that KLF4 may have a tumor suppressor effect. Indeed, the levels of KLF4 mRNA are significantly reduced in intestinal adenomas in the ApcMIN/+ mice and familial adenomatous polyposis (FAP) patients when compared to matched normal tissues 22. A similar reduction in KLF4 mRNA levels is seen in colorectal cancer specimens when compared to matched normal colonic tissues 23. Moreover, there is evidence for loss of heterozygosity (LOH) in the KLF4 locus in a subset of colorectal cancer specimens 23. Conversely, re-expression of KLF4 in a colorectal cancer cell line results in diminished tumorigenecity 24. Collectively, these findings indicate that KLF4 is a potential tumor suppressor in colorectal cancer. A similar down-regulation and growth suppressive effect of KLF4 has also been described in bladder cancer 25.

In contrast to the growth inhibitory effect of KLF4, KLF5 has a growth promoting effect in cultured cells based on a number of studies. The level of KLF5 mRNA is low in serum-deprived, quiescent NIH3T3 cells, but is acutely and significantly increased upon addition of serum or phorbol ester 26. Constitutive expression of KLF5 in NIH3T3 26 and intestinal epithelial cells, IEC6 27, results in an increased rate of proliferation and, eventually, a transformed phenotype as characterized by anchorage-independent growth 26 (Chanchevalap and Yang, unpublished observations). The level of KLF5 transcript is also elevated in NIH3T3 cells transformed by oncogenic H-Ras, and this increase mediates the transforming effect of oncogenic H-Ras 28. Fig. 2 illustrates the mechanism by which KLF5 mediates this growth-promoting function of oncogenic H-Ras. The increase in KLF5 expression is a direct result of Egr1, which is activated by mitogen-activated protein kinase (MAPK), which is itself stimulated by oncogenic H-Ras. The increase in KLF5 then stimulates expression of the genes encoding cyclin D1 28, cyclin B1, and Cdc2 (Nandan and Yang, unpublished observations), a change which culminates in accelerated cell proliferation and subsequent transformation. The importance of KLF5 in mediating cell proliferation is further demonstrated by the recent finding that the inhibitory effect of all-trans retinoic acid (ATRA) on proliferation of the intestinal epithelial cells, IEC6, is mediated by suppression of KLF5 expression 27.

Figure 2
figure 2

KLF5 is central in mediating the transforming effect of oncogenic H-Ras. The sequence of events from oncogenic H-Ras to KLF5 and subsequent target gene expression is shown. MAPK is mitogen-activated protein kinase. See reference 28 for detail. The effect of KLF5 on cyclin B1/Cdc2 expression is unpublished.

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KLF4 and KLF5 exert distinct physiologic functions in vivo as revealed by gene knockout experiments

Transgenic experiments involving targeted deletion of Klf4 and Klf5 genes in mice have demonstrated that the two genes exhibit distinct physiologic functions in vivo. Klf4 is normally expressed in the differentiating layers of the epidermis. Klf4−/− mice die shortly after birth and exhibit a defect in skin barrier function 29. Conversely, ectopic overexpression of Klf4 in the epidermis results in accelerated formation of the skin permeability barrier 30. Additional studies have revealed that the critical role of KLF4 in maintaining barrier function is probably based on its ability to coordinately regulate gene clusters that are involved in such function including the Sprr 31 and keratin families 20. KLF4 is also needed for terminal differentiation of goblet cells in the colon. Klf4−/− mice demonstrate a 90% reduction in the number of colonic goblet cells, as well as abnormal expression of goblet cell-specific markers 32.

Klf5−/− mice are embryonic lethal, but experiments using Klf5+/− mice showed that KLF5 is an important mediator of cardiovascular remodeling upon external stress. KLF5 is normally markedly induced in activated vascular smooth muscle cells and fibroblasts. In response to external stress such as injury induced by a vascular cuff, Klf5+/− mice showed diminished levels of arterial wall thickening, angiogenesis, cardiac hypertrophy and interstitial fibrosis 33. This physiologic effect of KLF5 is in part mediated by its ability to activate expression of genes encoding platelet-derived growth factor-A (PDGF-A) and transforming growth factor-β (TGF-β) 33.

Can KLF4 and KLF5 reverse their biological behavior in certain tumors?

The studies above demonstrate that KLF4 and KLF5 exhibit tumor suppressor and oncogenic activities, respectively, in a number of experimental systems. Can their effects be reversed in different settings? Indeed, several studies have indicated that they can. For example, expression of KLF4 is increased in dysplastic oral squamous epithelium 34. KLF4 mRNA and protein levels are also increased during progression of breast cancer 35, and nuclear localization of KLF4 is associated with an aggressive phenotype in early stage breast cancer 36. In contrast to the pro-proliferative effect of KLF5 shown above, loss of KLF5 expression has been observed in prostate 37 and breast cancer 38. Moreover, KLF5 has been shown to reduce colony formation in transformed intestinal epithelial cells 39. Therefore it appears that the biological behavior of KLF4 and 5 may change in the context of different tumor models, although the reasons for this are not well understood.

CONCLUSIONS

KLF4 and KLF5 are two members of the Krüppel-like factor family of transcription factors that exert important biological effects on cellular proliferation and differentiation in vivo and in vitro. Despite a close homology and similar developmental and tissue patterns of expression, the two KLFs exert very different, often opposing, effects on regulation of gene transcription and cellular proliferation. Both proteins also play a significant role in the process of tumorigenesis. Further characterization of KLF4 and 5 may advance the understanding of the molecular mechanisms regulating cellular proliferation and tumor formation.