Role of IGF-1R/IR signaling in epidermal development and morphogenesis

The epidermis protects the organism from external challenges and from dehydration. The establishment, maintenance and restoration of this epithelial barrier are driven by a balance between self-renewal and differentiation of interfollicular epidermal (IFE) progenitor cells. However, the upstream signals that regulate this balance are largely unknown. The major aim of this thesis was to identify and characterize the in vivo role of epidermal insulin/IGF signaling, which have been identified as key regulators of growth, metabolism and stem cell behavior in other tissues. Inactivating the insulin receptor (IR-/-), the IGF-1 receptor (IGF-1R-/-) or both (dko) specifically in the epidermis resulted in a progressive decrease in the number of suprabasal layers, first obvious at E16.5, although differentiation and apoptosis were unaltered. Instead, the phenotype was directly associated with a strong decrease in proliferative potential in vitro and altered IFE progenitor cell behavior in vivo. Furthermore, rescue experiments identified the small GTPase Rac1 as a key target of IGF-1R/IR signaling that regulates epidermal morphogenesis in vivo and proliferative potential in vitro. To address how IR/IGF-1R regulates epidermal morphogenesis, we focused on E16.5 dko embryos, when the phenotype is first obvious. Whereas no change was observed in expression of the proliferation marker Ki67, a reduced number of anaphase spindles and an increased number of metaphase spindles were found in dko mice compared to control, and in vitro in cultured keratinocytes. This indicated an arrest in the spindle check point of the cell cycle in the absence of IR/IGF-1R. This was associated with disturbed spindle formation in IGF-1R-/- metaphase cells and reduced expression of AuroraB, a key regulator of mitosis, as well as other cell cycle regulators, such as p53 and stratifin (14-3-3σ). Interestingly, the reduction in cell divisions upon loss of IR/IGF-R was predominantly on the expense of asymmetric divisions and correlated with reduced p63 expression, a known regulator of this process. Since asymmetric cell divisions have been implicated in regulating epidermal stratification, the results indicate that epidermal Insulin/IGF1 signaling couples cell cycle progression to asymmetric divisions thereby regulating the number of suprabasal layers in the IFE during morphogenesis. In short, the results in this thesis have identified epidermal IR/IGF-1R as key regulators of epidermal morphogenesis, proliferative potential, IFE progenitor cells and asymmetric cell divisions

Insulin/IGF-1 Controls Epidermal Morphogenesis via Regulation of FoxO-Mediated p63 Inhibition

The multilayered epidermis is established through a stratification program, which is accompanied by a shift from symmetric toward asymmetric divisions (ACD), a process under tight control of the transcription factor p63. However, the physiological signals regulating p63 activity in epidermal morphogenesis remain ill defined. Here, we reveal a role for insulin/IGF-1 signaling (IIS) in the regulation of p63 activity. Loss of epidermal IIS leads to a biased loss of ACD, resulting in impaired stratification. Upon loss of IIS, FoxO transcription factors are retained in the nucleus, where they bind and inhibit p63-regulated transcription. This is reversed by small interfering RNA-mediated knockdown of FoxOs. Accordingly, transgenic expression of a constitutive nuclear FoxO variant in the epidermis abrogates ACD and inhibits p63-regulated transcription and stratification. Collectively, the present study reveals a critical role for IIS-dependent control of p63 activity in coordination of ACD and stratification during epithelial morphogenesis

Persistent transcription-blocking DNA lesions trigger somatic growth attenuation associated with longevity

The accumulation of stochastic DNA damage throughout an organism's lifespan is thought to contribute to ageing. Conversely, ageing seems to be phenotypically reproducible and regulated through genetic pathways such as the insulin-like growth factor-1 (IGF-1) and growth hormone (GH) receptors, which are central mediators of the somatic growth axis. Here we report that persistent DNA damage in primary cells from mice elicits changes in global gene expression similar to those occurring in various organs of naturally aged animals. We show that, as in ageing animals, the expression of IGF-1 receptor and GH receptor is attenuated, resulting in cellular resistance to IGF-1. This cell-autonomous attenuation is specifically induced by persistent lesions leading to stalling of RNA polymerase II in proliferating, quiescent and terminally differentiated cells; it is exacerbated and prolonged in cells from progeroid mice and confers resistance to oxidative st

Epidermal insulin/IGF-1 signalling control interfollicular morphogenesis and proliferative potential through Rac activation

The lifelong self-renewal of the epidermis is driven by a progenitor cell population with high proliferative potential. To date, the upstream signals that determine this potential have remained largely elusive. Here, we find that insulin and insulin-like growth factor receptors (IR and IGF-1R) determine epidermal proliferative potential and cooperatively regulate interfollicular epidermal morphogenesis in a cell autonomous manner. Epidermal deletion of either IR or IGF-1R or both in mice progressively decreased epidermal thickness without affecting differentiation or apoptosis. Proliferation was temporarily reduced at E17.5 in the absence of IGF-1R but not IR. In contrast, clonogenic capacity was impaired in both IR- and IGF-1R-deficient primary keratinocytes, concomitant with an in vivo loss of keratin 15. Together with a reduction in label-retaining cells in the interfollicular epidermis, this suggests that IR/IGF-1R regulate progenitor cells. The expression of dominant active Rac rescued clonogenic potential of IR/IGF-1R-negative keratinocytes and reversed epidermal thinning in vivo. Our results identify the small GTPase Rac as a key target of epidermal IR/IGF-1R signalling crucial for proliferative potential and interfollicular morphogenesis