Ca2+ and BMP-6 Signaling Regulate E2F during Epidermal Keratinocyte Differentiation

Abstract

The epidermis consists of a squamous epithelium continuously replenished by committed stem cells, which can either self-renew or differentiate. We demonstrated previously that E2F genes are differentially expressed in developing epidermis (Dagnino, L., Fry, C. J., Bartley, S. M., Farnham, P., Gallie, B. L., and Phillips, R. A. (1997) Cell Growth Differ. 8, 553-563). Thus, we hypothesized that various E2F proteins likely play distinct growth regulatory roles in the undifferentiated stem cells and in terminally differentiated keratinocytes. To further understand the function of E2F genes in epidermal morphogenesis, we have examined the expression, regulation, and protein-protein interactions of E2F factors in undifferentiated cultured murine primary keratinocytes or in cells induced to differentiate with Ca 2؉ or BMP-6 (bone morphogenetic protein 6). We find similar patterns of E2F regulation with both differentiating agents and demonstrate a switch in expression from E2F-1, -2, and -3 in undifferentiated, proliferating cells to E2F-5 in terminally differentiated keratinocytes. Inhibition of keratinocyte proliferation by transforming growth factor-␤1 did not enhance E2F-5 protein levels, suggesting that this response is specific to differentiation rather than reversible cell cycle withdrawal. E2F-5 up-regulation is also accompanied by formation of heteromeric nuclear complexes containing E2F5, p130, and histone deacetylase (HDAC) 1. Overexpression of E2F5 specifically inhibited DNA synthesis in undifferentiated keratinocytes in an HDAC-dependent manner, suggesting that E2F-5⅐p130⅐HDAC1 complexes are likely involved in the permanent withdrawal from the cell cycle of keratinocytes responding to differentiation stimuli.The skin epithelium, or epidermis, provides a barrier between the internal and external regions of the body, is constantly subjected to physical and chemical stress, and consequently has high renewal rates. The epidermis consists of a stratified squamous epithelium composed mainly of keratinocytes at different stages of differentiation (1, 2). Within the epidermis, the basal cell layer is attached to a basement membrane (3-5), is closest to the dermis, and contains the stem cells. These cells have continuous self-renewal potential and are responsible for renewing and maintaining the epithelium (6). Committed basal cells lose their proliferative capacity, detach from the basement membrane, and initiate terminal differentiation. Differentiating cells migrate upwards and form postmitotic suprabasal skin layers. The signals and intracellular networks that dictate changes in keratinocyte proliferation and differentiation are poorly understood. These networks are extremely important, because they ultimately determine proper skin morphogenesis and homeostasis.Important cellular networks that regulate proliferation and differentiation in a variety of cell types include cyclins, pRb family proteins and E2F factors. The E2F family of transcription factors consists of six known genes that form heterodimers with D...

Phosphorylation by p38 MAP kinase is required for E2F1 degradation and keratinocyte differentiation

“…Involvement of p38 MAPK in E2F1 phosphorylation in keratinocytes E2F1 is a phosphoprotein in a variety of cell types, including primary murine epidermal keratinocytes (D'Souza et al, 2001). In these cells, differentiation is associated with additional post-translational events that regulate both endogenous and exogenously expressed E2F1, including p38b MAPK-dependent induction of nuclear export and proteolysis (Ivanova et al, 2006;.…”

“…Primary mouse keratinocytes isolated from 1-to 2-day-old CD-1 mice were cultured in Ca 2 þ -free Eagle's minimum essential medium (EMEM) (06-174G, Lonza, Rockland, ME, USA) and supplements (low Ca 2 þ medium) as described earlier (D'Souza et al, 2001). Keratinocytes were induced to differentiate by increasing the Ca 2 þ concentration in the growth medium to 1 mM (high Ca 2 þ medium).…”

“…The complex nature of the epidermis and the differentiation programs present in keratinocytes necessitate the activation of multiple mechanisms to modulate E2F activity and specificity, including mRNA and protein expression, as well as changes in subcellular localization (Dagnino et al, 1997;D'Souza et al, 2001;Apostolova et al, 2002). In particular, differentiation stimuli in keratinocytes activate signaling pathways that modulate E2F1 post-translationally (Ivanova et al, 2006;.…”

Unique and overlapping functions of pRb and p107 in the control of proliferation and differentiation in epidermis

“…We and others have found a role for Rb family members during the in vitro differentiation of keratinocytes (Martinez et al, 1999;Paramio et al, 1998), involving the specific interaction with distinct E2F factors (D'Souza et al, 2001;Paramio et al, 2000). However, little is known about the function of this family during normal skin development and epidermal homeostasis in vivo.…”

E2F1 stability is regulated by a novel-PKC/p38β MAP kinase signaling pathway during keratinocyte differentiation

“…E2F1, -2 and -3 are abundant in undifferentiated keratinocytes. Differentiation triggers downregulation of these E2F forms and upregulation of E2F5 (D'Souza et al, 2001). The biological importance of E2F1 function and regulation in epidermal homeostasis has been amply demonstrated.…”