Cardiovascular disease (CVD) is the leading cause of illness and death in the United States and the major cause of mortality and disability in patients with diabetes mellitus.29, 30
It has been documented that KLF11 is a diabetes relevant gene.11, 31-33
Indeed, recent research reveals that alterations in the KLF11 pathway impact the development of neonatal and juvenile diabetes.10
Here, the observation that KLF11 is highly expressed in ECs and that its expression could be induced by TNF-α prompted us to address the relationship between KLF11 and EC activation.
The recruitment of leukocytes to ECs, an early stage of inflammatory diseases such as atherosclerosis, is mediated by the endothelial expression of adhesion molecules such as ICAM-1, VCAM-1 and E-selectin.4
Through in vitro
gain- and loss-of-function approaches, we demonstrate that KLF11 overexpression potently inhibits TNF-α-induced expression of adhesion molecules in endothelial cells while, in response to inflammatory stimuli, THP-1 cells show increased adhesion to si-KLF11-treated HUVECs concomitant with increased levels in VCAM-1 and E-selectin in those cells. Furthermore, a more fundamental contribution to our understanding KLF11 function in the present study is provided by the finding that KLF11 deficiency in vivo
exacerbates the rolling and adhesion of leukocytes on endothelial cells in an LPS-induced animal model of endothelial dysfunction. The conventional KLF11-/-
mice used here also lack KLF11 in the leukocytes. Our observation of increased adhesion of THP-1 (wild type for endogenous KLF11) to si-KLF11-treated HUVECs in vitro
argues that this effect is mostly driven by the exacerbated activation of the endothelium in the KLF11-/-
mice resulting in enhanced expression of adhesion molecules. Taken together, our study provides evidence that KLF11 has a potent anti-inflammatory effect on vascular ECs and is critical for modulation of leukocytes-EC interaction.
KLF11, a nuclear-located transcription factor, regulates target genes through binding to a consensus sequence present in their promoters.5
We investigated the transcriptional mechanism underlying the anti-inflammatory effect of KLF11. We found that although KLF11 does not significantly regulate the pro-inflammatory adhesion molecules and cytokines under basal conditions, its effects are evident in response to TNF-α and other stimuli. NF-κB plays a critical mediator role in response to most inflammatory stimuli in various cell lines and in ECs in particular.34, 35
In the present study, we demonstrate that KLF11 inhibits NF-κB signaling via
a physical interaction with NF-κB p65. CHIP assay data further showed that depletion of KLF11 by RNAi exacerbates the binding of p65 to the VCAM-1 and E-selectin promoters in ECs, thereby confirming that KLF11 regulation of adhesion molecules at the level of transcription involves negative regulation of the NF-κB pro-inflammatory response. The repressor domains R1, R2, R3 and the zinc fingers of KLF11 bind independently to multiple chromatin remodelers to fulfill its functions.22, 23, 33, 36
Noteworthy, studies looking at genetic variations in the KLF11 gene in distinct human populations revealed a variant (Ala347Ser) within R3 domain that segregates with diabetes in families with early-onset type 2 diabetes.31
Here, we demonstrate that KLF11 R3-ZNF could inhibit the p65-induced transcription of the VCAM-1 promoter () and the R3-ZNF physically binds to p65 (). In fact, our data suggest that this specific repression domain of KLF11 is sufficient to regulate pro-inflammatory adhesion molecules expression in response to TNF-α in EC and demonstrate that NF-κB, a good example of regulatory pathways of EC inflammation, is at least one of the most important signaling molecules mediating this effect. Actually, many inflammatory mediators such as activator protein-1 (AP-1), nuclear factor of activated T-cells (NFAT), and Ets137, 38
are involved in the transduction of extracellular pro-inflammatory stimuli to intracellular signaling pathways. Based on the data from the present study, we cannot completely rule out that KLF11 might inhibit EC inflammation via
multiple signaling pathways under conditions of pro-inflammatory stimulation. Nevertheless, the data here reported provides biochemical and cell biological evidence demonstrating that KLF11 is upregulated in response to inflammatory stimuli and inhibits EC inflammation via
inhibition of p65. KLF11 possibly acts in a negative-feedback manner to ensure the optimal p65 activation levels thus maintaining the pro- and anti-inflammatory homeostatic balance, which is required for proper “physiological” inflammatory responses.
It has been documented that KLFs 2, 4 and 6 are expressed in ECs and have important roles in EC biology. KLF6 is induced after vascular injury and stimulates endogenous endoglin expression in vascular repair.39
KLF2 and KLF4 can be induced by laminar shear stress in ECs.40, 41
However, KLF2 is downregulated while KLF4 is increased in TNF-α-stimulated ECs.20
Both KLF2 and KLF4 suppress TNF-α-induced EC activation evidenced by decreased expression of pro-inflammatory adhesion molecules.20,19, 42
Our data indicates that the anti-inflammatory effects of KLF11 on EC cannot be explained by KLF11 interfering with KLF2 or KLF4 since we found that changes in KLF11 expression do not translate in changes in the expression of KLF2 or KLF4 either in vitro
or in vivo
and KLF11 inhibitory effect on the expression of EC adhesion molecules is not affected by KLF2 or KLF4 knockdown in ECs. The synergistic effects observed between KLF11 and KLF2 or KLF4 on the VCAM-1 and E-selectin expression may be a result of a concomitant inhibition of the NF-κB transcriptional activity resulting from sequestration of its co-transcriptional activator CBP/p30043
or the concurrent inhibition of NF-κB pathway by KLF11, possibilities that remain to be addressed. More interestingly, we demonstrate that KLF11 may have broader negative effects on adhesion molecules, since KLF11 potently inhibits ICAM-1 expression while KLF2 does not.19
A number of studies have demonstrated that the ICAM-1 promoter contains transcription factor binding sites such as NF-κB, AP-1, ets-1, SP1, STAT, and PKC-zeta.44, 45
KLF2 may activate some of these transcription factors thus resulting in a distinct, overall non-inhibitory effect for KLF2 overexpression on the ICAM-1 promoter.
In summary, here we demonstrate for the first time an important homeostatic role of KLF11 as an anti-inflammatory factor controlling leukocytes recruitment via modulation of physiological responses in the expression of pro-inflammatory adhesion molecules in ECs. This knowledge extends the current understanding of how KLF11 regulate key cellular functions in the vascular system thereby helping to further underscore the important role of this transcription pathway in maintaining EC homeostasis. KLF11 is thus a potential molecular target for treatment of EC inflammation-associated cardiovascular diseases such as atherosclerosis and diabetic vascular pathologies.