E3 Ubiquitin Ligases Regulate Stem Cell Functions

Stem cells require a tightly controlled intracellular signaling network to regulate fate decisions. Imbalances in self-renewal and commitment towards specialized cell types will compromise tissue integrity. Disturbances in the regulatory program that controls self-renewal have been associated with carcinogenesis (Orford and Scadden, 2008). Post-translational modifications play an essential role in the control of stem cell functions (Werner et al., 2017).

Regulation through ubiquitination has been proven in embryonic as well as in adult stem cells (Strikoudis et al., 2014). Multiple mechanisms by which ubiquitination affects stem cell fate decisions have emerged (Werner et al., 2017). Stem cells need to adjust their program to local and systemic demands. Information will be provided through secreted molecules or cell-to-cell contact within specialized stem cell niches (Orford and Scadden, 2008). Localization and stability of receptor complexes can be regulated through ubiquitination for appropriate integration of extracellular signals (Werner et al., 2017). Downstream effector proteins can be altered in their activation state, binding partner affinity or stability via ubiquitin tags. Ubiquitin ligases further target transcription factors as well as cell cycle regulators. The latter process has been proven crucial for stem cell quiescence and thus maintenance of the systemic stem cell pool (Werner et al., 2017). Moreover, epigenetic mechanisms play a role in stem cell fate decisions and are particularly investigated in cell reprogramming, induced pluripotent stem cells (iPS cells) and carcinogenesis (Strikoudis et al., 2014). Ubiquitination of histones can lead to transcriptional activation as well as transcriptional repression. It may also result in subsequent chromatin modifications (Strikoudis et al., 2014).

1.4.1 The E3 Ubiquitin Ligase Itch in Stem Cells.

To date, the E3 ubiquitin ligase Itch has been primarily studied within the immune system and especially in the regulation of T cells (Aki et al., 2015). Itch protein is expressed in a wide range of different tissues and cells types, though. More recently, its role in the control of stem and progenitor cell populations has been discovered (Aki et al., 2015).

Liao et al. studied the function of Itch in embryonic stem cells (ES cells) through a loss-of-function approach (Liao et al., 2013). Down-regulation of Itch via sh-RNA resulted in decreased self-renewal in a murine ES cell line. The authors further demonstrated that Itch expression levels increased upon induction of somatic cell reprogramming. Knock-down of Itch diminished the yield of iPS clones following transduction with four reprogramming transcription factors (Oct4, Sox2, Klf4, cMyc).

The positive regulation of ES cell self-renewal and induced somatic reprogramming was attributed to the interaction of Itch with the transcription factor Oct4 (Liao et al., 2013). Oct4 is considered a core regulatory component of ES cell self-renewal (Jaenisch and Young, 2008). Oct4 ubiquitination through Itch in ES cells led to increased transcriptional activity (Liao et al., 2013).

In contrast to the positive effect of Itch on ES cells, Rathinam et al. identified Itch as a negative regulator of hematopoietic stem cell self-renewal (Rathinam et al., 2011).

The Itch knockout mouse showed increased frequencies of hematopoietic stem and progenitor cells, including long term HSCs. Itch-deficient HSCs hyperproliferated and exhibited an augmented repopulation activity in serial and competitive transplantation assays. The loss of Itch led to increased Notch1 signaling. Itch ubiquitinated Notch1 in HSCs and downregulation of Notch signaling in the absence of Itch partially rescued the HSC phenotype in vitro and in vivo (Rathinam et al., 2011).

Several studies have investigated the role of Itch in skin regeneration and wound healing (Rossi et al., 2006; Giamboi-Miraglia et al., 2015). Rossi et al. found that p63 ubiquitination through Itch in keratinocytes resulted in p63 degradation (Rossi et al., 2006). Itch levels increased whereas dNp63 levels concomitantly decreased during human and mouse keratinocyte differentiation. P63 has been established as a key regulator in the development and regeneration of different epithelial tissues and dNp63 is vital for basal layer formation (Rossi et al., 2006). It was further demonstrated that Itch-deficient keratinocytes hyperproliferated in vivo and that Itch knockout mice exhibited a thickened basal layer at developmental stages and as newborns (Giamboi-Miraglia et al., 2015). This hyperplasia was not present in adult mice. Itch-deficient skin further exhibited superior wound healing capacities. The absence of Itch resulted in increased p63, cJun and JunB protein levels as well as increased Notch signaling in keratinocytes (Giamboi-Miraglia et al., 2015).

Together, these results suggested that Itch negatively regulated stem cells within the skin basal layer.

1.4.2 Mesenchymal Stem Cell Regulation Through E3 Ubiquitin Ligases.

The molecular circuits that control hematopoietic stem cell functions have been most intensively investigated as compared with other tissues. A variety of E3 ligases are currently known to regulate HSC quiescence, self-renewal and differentiation (Strikoudis et al., 2014). Genetic mouse models sharpened our understanding of these regulatory mechanisms and established the basis for clinical applications (Orford and Scadden, 2008). Though HSCs serve as a paradigm for adult stem cell biology, the function of particular E3 ligases need to be investigated and interpreted independently in other stem cells (Strikoudis et al., 2014).

A detailed understanding of the molecular network that guides MSC functions is just beginning to emerge. In contrast to HSCs, few E3 ligases have been identified to regulate MSC self-renewal and differentiation through direct in vivo identification of the stem cell subset:

The von Hippel Lindau protein (VHL) is an E3 ubiquitin ligase and a critical negative regulator of hypoxia inducible factors (HIF) (Mangiavini et al., 2015). The Vhl-Hif axis has been investigated at multiple steps of skeletogenesis during development as well as in postnatal osteoblast and chondrocyte differentiation. Postnatally, Vhl seemed to positively regulate early chondroblast commitment and to restrict osteoblast differentiation in a Hif-dependent manner (Mangiavini et al., 2014; Weng et al., 2014). Interestingly, Prx1-Cre-driven conditional deletion of Vhl led to fibrosis and mesenchymal soft tissue tumors (Mangiavini et al., 2015). These findings were not described when Vhl was deleted at a later stage of osteoblast or chondrocyte commitment (Weng et al., 2014; Mangiavini et al., 2015). A detailed molecular signature of the vimentin and S100 positive, hyperproliferating, fibroblastic cells has not been described. The soft tissue tumor development was dependent on the Hif-1α-inducible matrix protein Ctgf (connective tissue growth factor) (Mangiavini et al., 2015).

The E3 ligase Cbl was studied in early osteoblast commitment using the CblY737F knock-in mouse (Scanlon et al., 2017). Through a point mutation, the Cbl-Pi3k

interaction is abrogated. In a fracture model, CblY737F mice exhibited periosteal thickening with relative accumulation of Osx-RFP positive progenitors. This resulted in enhanced osteogenic differentiation and ossification of the fracture (Scanlon et al., 2017).