Research paper
Functional analysis of ZFP36 proteins in keratinocytes

https://doi.org/10.1016/j.ejcb.2016.04.007Get rights and content

Abstract

The ZFP36 family of zinc finger proteins, including ZFP36, ZFP36L1, and ZFP36L2, regulates the production of growth factors and cytokines via destabilization of the respective mRNAs. We could recently demonstrate that in cultured keratinocytes, expression of the ZFP36, ZFP36L1, and ZFP36L2 genes is induced by growth factors and cytokines and that ZFP36L1 is a potent regulator of keratinocyte VEGF production.

We now further analyzed the localization and function of ZFP36 proteins in the skin, specifically in epidermal keratinocytes.

We found that in human epidermis, the ZFP36 protein could be detected in basal and suprabasal keratinocytes, whereas ZFP36L1 and ZFP36L2 were expressed mainly in the basal layer, indicating different and non-redundant functions of the three proteins in the epidermis.

Consistently, upon inhibition of ZFP36 or ZFP36L1 expression using specific siRNAs, there was no major effect on expression of the respective other gene. In addition, we demonstrate that both ZFP36 and ZFP36L1 influence keratinocyte cell cycle, differentiation, and apoptosis in a distinct manner. Finally, we show that similarly as ZFP36L1, ZFP36 is a potent regulator of keratinocyte VEGF production. Thus, it is likely that both proteins regulate angiogenesis via paracrine mechanisms.

Taken together, our results suggest that ZFP36 proteins might control reepithelialization and angiogenesis in the skin in a multimodal manner.

Introduction

Cutaneous wound repair is a highly complex process involving many different cell types, such as keratinocytes, fibroblasts, endothelial cells, and immune cells. The different stages of healing—inflammation, reepithelialization, contraction and angiogenesis—are regulated by a multitude of growth factors and cytokines, such as EGF, TGFβ, FGF1, KGF, HGF, VEGF, and TNFα.

The epidermis, which forms the outer layer of the skin, consists mainly of keratinocytes. They differentiate from the basal layer, however, during wound healing, epidermal keratinocytes migrate into the wound site and become hyperproliferative (reviewed by Martin, 1997, Gurtner et al., 2008).

ZFP36 (tristetraprolin TTP, Tis11, Nup34), ZFP36L1 (Tis11B, ERF1, BRF1, berg36), and ZFP36L2 (Tis11D, ERF2, BRF2) are members of the ZFP36 family of RNA-binding zinc finger proteins (DuBois et al., 1995, Varnum et al., 1991). They appear to be evolutionary “old” proteins, since ZFP36 proteins have been identified in a broad variety of species from yeast (Ma and Herschman, 1995) to humans (Taylor et al., 1991), except for the ZFP36L3 isoform, which seems to be mouse-specific (Blackshear et al., 2005).

ZFP36 proteins bind to and destabilize specific target mRNAs, such as the TNFα, the GM-CSF, and the VEGF transcripts, thus reducing expression of the respective genes. Recognition occurs via binding of the ZFP36 proteins to AU-rich elements (AREs) within the target transcripts’ 3′untranslated regions (3′UTRs) (Lai et al., 2000, Carballo et al., 2000, Ciais et al., 2004; for review, see Baou et al., 2009, Sanduja et al., 2011, Ciais et al., 2013).

Despite the fact that AREs were first discovered in cytokine and protooncogene sequences (Caput et al., 1986), genome-wide screens have identified AREs in a broad variety of different mRNAs encoding proteins of multiple and diverse functions (Bakheet et al., 2001, Lai et al., 2006).

Expression of the ZFP36/Zfp36 (human/rodent), ZFP36L1/Zfp36l1 and ZFP36L2/Zfp36l2 genes can be induced by various stimuli, e.g. growth factors, in a broad range of cell types (Varnum et al., 1989a, Varnum et al., 1989b, Gomperts et al., 1992, Reppe et al., 2004, Manabe et al., 1999). All three genes are expressed in a huge variety of cell types and tissues, however, each has its unique and distinct spatial and temporal expression pattern (Carrick and Blackshear, 2007). With respect to the physiological role of the ZFP36 proteins under in vivo conditions, studies in knock-out mice demonstrated roles for ZFP36, ZFP36L1, and ZFP36L2, in inflammation, chorioallantoic fusion and vascularization, and early embryonic development, respectively (Taylor et al., 1996, Stumpo et al., 2004, Ramos et al., 2004, Bell et al., 2006). In addition, ZFP36L1 has been implicated in myogenesis (Busse et al., 2008), and ZFP36 has been identified as regulator of dendritic cell maturation (Emmons et al., 2008), but little is known so far about a possible role of ZFP36 proteins in keratinocytes or in cutaneous wound repair.

We could recently demonstrate induction of all three ZFP36 genes, specifically of ZFP36 and ZFP36L1, after scratch-wounding in vitro. In addition, we could show that ZFP36L1 regulates keratinocyte VEGF production, thereby potentially modulating the angiogenic response in wound tissue (Hacker et al., 2010). Furthermore, most interestingly, a recent report demonstrated that the anti-inflammatory effect of glucocorticoids on the epidermis is in part mediated by ZFP36 (Sevilla et al., 2015).

Now, we functionally analyzed the role of the three ZFP36 proteins in epidermal keratinocytes. Specifically, we demonstrate that each of the three genes has its specific and unique spatial expression pattern in the epidermis and that inhibiting ZFP36 or ZFP36L1 expression in cultured keratinocytes induces apoptosis, blocks cell proliferation, and enhances VEGF production. Our data indicate that ZFP36 proteins might be multi-functional players in the skin and perhaps also in wound healing.

Section snippets

Skin tissue

Paraffin sections from human belly skin were purchased from Zyagen, San Diego, CA, USA.

Cell culture

Human HaCaT keratinocytes were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum.

Scratch-wounding

HaCaT cells were grown in 6-well plates and serum-starved overnight. They were then scratched with a scalpel to yield wounds with uniform width.

Transfection of keratinocytes with siRNA

1 × 106 HaCaT keratinocytes per well were trypsinized, washed, resuspended and reverse-transfected with ZFP36-, or ZFP36L1-specific siRNA or an unspecific

Localization of ZFP36, ZFP36L1, and ZFP36L2 in the skin

In our previous study (Hacker et al., 2010), we could demonstrate induction of all three ZFP36 genes after scratch-wounding of human HaCaT keratinocytes in vitro. In addition, after wounding of murine skin in vivo, we could detect upregulation of ZFP36 expression. However, still, the spatial expression patterns of the three ZFP36 genes in the skin remained unclear, specifically, it was not known whether the genes were expressed in the dermal or the epidermal compartment or both.

Thus, we aimed

Discussion

When studying the localization of ZFP36, ZFP36L1, and ZFP36L2 in human and murine skin, we found that all three proteins were present in both the epidermal the dermal compartments. Nevertheless, intra-epidermally, the localization of ZFP36 differed from that of the other two isoforms: Whereas ZFP36L1 and ZFP36L2 could be detected in almost all cells and particularly in the basal, proliferating layer, ZFP36 could be found in approximately 50% of the cells all over the epidermis, both in basal

Acknowledgements

We thank Sabine Werner, ETH Zurich, for RNA and tissue sections. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Mu 1556/5-1) (to B.M.).

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