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The IGF-1/IGF-1R signaling axis in the skin: a new role for the dermis in aging-associated skin cancer.

Lewis DA, Travers JB, Somani AK, Spandau DF - Oncogene (2009)

Bottom Line: In human skin, epidermal keratinocytes do not express IGF-1, and hence the IGF-1 receptor on keratinocytes is activated by IGF-1 secreted from dermal fibroblasts.Finally, the appropriate UVB response is restored in geriatric skin in vivo through pretreatment with exogenous IGF-1.These studies provide further evidence for a role of the IGF-1 receptor (IGF-1R) in suppressing UVB-induced carcinogenesis, suggest that fibroblasts have a critical role in maintaining appropriate activation of the keratinocyte IGF-1R, and imply that reduced expression of IGF-1 in geriatric skin could be an important component in the development of aging-related non-melanoma skin cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.

ABSTRACT
The appropriate response of human keratinocytes to ultraviolet-B (UVB) is dependent on the activation status of the insulin-like growth factor 1 (IGF-1) receptor. Keratinocytes grown in conditions in which the IGF-1 receptor is inactive inappropriately replicate in the presence of UVB-induced DNA damage. In human skin, epidermal keratinocytes do not express IGF-1, and hence the IGF-1 receptor on keratinocytes is activated by IGF-1 secreted from dermal fibroblasts. We now show that the IGF-1 produced by human fibroblasts is essential for the appropriate UVB response of keratinocytes. Furthermore, the expression of IGF-1 is silenced in senescent fibroblasts in vitro. Using quantitative reverse transcriptase-PCR and immunohistochemisty, we can show that IGF-1 expression is also silenced in geriatric dermis in vivo. The diminished IGF-1 expression in geriatric skin correlates with an inappropriate UVB response in geriatric volunteers. Finally, the appropriate UVB response is restored in geriatric skin in vivo through pretreatment with exogenous IGF-1. These studies provide further evidence for a role of the IGF-1 receptor (IGF-1R) in suppressing UVB-induced carcinogenesis, suggest that fibroblasts have a critical role in maintaining appropriate activation of the keratinocyte IGF-1R, and imply that reduced expression of IGF-1 in geriatric skin could be an important component in the development of aging-related non-melanoma skin cancer.

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IGF-1 secreted by normal human fibroblasts can protect keratinocytes from UVB-induced apoptosis(A) To determine if normal human fibroblasts can produce a protective factor which can activate the IGF-1R and restore the normal UVB response of keratinocytes, normal human fibroblasts were grown in the EpiLife NoIn media for 48 h (referred to as Conditioned Media, CM). Keratinocytes were grown in EpiLife NoIn media for 48 h and subsequently the media was replaced with EpiLife Complete media, EpiLife NoIn media, or EpiLife NoIn-CM derived from fibroblasts. Aliquots of EpiLife Complete media and CM-NoIn were treated with a neutralizing antibody to IGF-1 (Calbiochem, α-IGF-1, Ab2; inhibits 50% at suggested concentration) 15 min prior to adding it to the keratinocytes. One hour following media exchange, the keratinocytes were irradiated with 0 or 400 J/m2 of UVB. Six hours post-irradiation, the keratinocytes were harvested and assayed for the induction of apoptosis. Error bars indicate the standard error of the mean; the asterisk indicates significant difference between caspase 3 specific activities derived from keratinocytes grown in Complete media versus NoIn media (p<0.01, t-test). The data presented represent three independent assays. (B and C) Fibroblasts were treated with IGF-1-specific siRNA or non-specific siRNA for 24 h. NoIn media was then added to the fibroblasts for 48 h. At that time, the media was removed, filter-sterilized, and added to keratinocyte cultures. (B) qRT-PCR analysis of IGF-1 expression in fibroblasts following the indicated siRNA treatment. Error bars indicate the standard error of the mean; asterisk indicates significant (p<0.02, t-test) difference from nonsense siRNA-treated keratinocytes. (C) Keratinocytes were grown in NoIn media for 48 h, then the indicated media was added for 1 h. At that time, the keratinocytes were irradiated with the indicated dose of UVB and harvested six hours post-irradiation for caspase 3 analysis. Error bars indicate the standard error of the mean; the asterisks indicate significant difference between caspase 3 specific activities derived from keratinocytes grown in NoIn media + !GF-1 (p<0.03, t-test). The data presented represent three independent assays.
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Figure 2: IGF-1 secreted by normal human fibroblasts can protect keratinocytes from UVB-induced apoptosis(A) To determine if normal human fibroblasts can produce a protective factor which can activate the IGF-1R and restore the normal UVB response of keratinocytes, normal human fibroblasts were grown in the EpiLife NoIn media for 48 h (referred to as Conditioned Media, CM). Keratinocytes were grown in EpiLife NoIn media for 48 h and subsequently the media was replaced with EpiLife Complete media, EpiLife NoIn media, or EpiLife NoIn-CM derived from fibroblasts. Aliquots of EpiLife Complete media and CM-NoIn were treated with a neutralizing antibody to IGF-1 (Calbiochem, α-IGF-1, Ab2; inhibits 50% at suggested concentration) 15 min prior to adding it to the keratinocytes. One hour following media exchange, the keratinocytes were irradiated with 0 or 400 J/m2 of UVB. Six hours post-irradiation, the keratinocytes were harvested and assayed for the induction of apoptosis. Error bars indicate the standard error of the mean; the asterisk indicates significant difference between caspase 3 specific activities derived from keratinocytes grown in Complete media versus NoIn media (p<0.01, t-test). The data presented represent three independent assays. (B and C) Fibroblasts were treated with IGF-1-specific siRNA or non-specific siRNA for 24 h. NoIn media was then added to the fibroblasts for 48 h. At that time, the media was removed, filter-sterilized, and added to keratinocyte cultures. (B) qRT-PCR analysis of IGF-1 expression in fibroblasts following the indicated siRNA treatment. Error bars indicate the standard error of the mean; asterisk indicates significant (p<0.02, t-test) difference from nonsense siRNA-treated keratinocytes. (C) Keratinocytes were grown in NoIn media for 48 h, then the indicated media was added for 1 h. At that time, the keratinocytes were irradiated with the indicated dose of UVB and harvested six hours post-irradiation for caspase 3 analysis. Error bars indicate the standard error of the mean; the asterisks indicate significant difference between caspase 3 specific activities derived from keratinocytes grown in NoIn media + !GF-1 (p<0.03, t-test). The data presented represent three independent assays.

Mentions: Proper function of the skin requires the synergistic interactions between dermal fibroblasts and epidermal keratinocytes. The proliferation, differentiation, and stress responses of keratinocytes are greatly altered when deficiencies in dermal fibroblasts occur. We hypothesize that the normal UVB-response (defined as protection from carcinogenic initiation) of keratinocytes is also highly dependent on the status of dermal fibroblasts. As described above, we have defined conditions where the appropriate UVB-response of normal human keratinocytes grown in tissue culture can be altered by differential activation of the IGF-1R. We believe these culture conditions that alter the UVB-response in keratinocytes in vitro reflect instances of deficient fibroblast function in vivo. To determine if normal human fibroblasts can produce a protective factor which can activate the IGF-1R and restore the normal UVB response of keratinocytes, normal human fibroblasts were grown in the EpiLife NoIn media for forty-eight hours (referred to as Conditioned Media, NoIn-CM) and then the NoIn-CM was removed from the fibroblasts and filter sterilized. Keratinocytes were grown in EpiLife NoIn media for 48 hours and subsequently the media was replaced with EpiLife Complete media, with NoIn media, or NoIn-CM from fibroblasts. One hour following media exchange, the keratinocytes were irradiated with 0 or 400 J/m2 of UVB and subsequently assayed for the induction of apoptosis. As seen in Figure 2A, UVB irradiation induced a modest induction of apoptosis in keratinocytes treated with EpiLife Complete media and substantial apoptosis in keratinocytes grown in NoIn media. However, keratinocytes treated with NoIn-CM were resistant to UVB-induced apoptosis, similar to those treated with EpiLife Complete media. These data indicate that normal human fibroblasts produced a diffusible factor into the EpiLife insulin-deficient media that protected the keratinocytes from UVB-induced apoptosis. To determine if the protective factor produced by fibroblasts was IGF-1, we added a neutralizing antibody specific to IGF-1 to the fibroblast NoIn-CM. Addition of the IGF-1 neutralizing antibody prevented complete protection of UVB-induced apoptosis (Figure 2A). As expected, the addition of the neutralizing IGF-1 antibody to EpiLife Complete media did not alter the keratinocyte UVB response indicating a lack of cross-reactivity to the insulin in the EpiLife Complete media by the anti-IGF-1 antibody. At the concentration of IGF-1 antibody used, the manufacturers (Calbiochem, α-IGF-1 Ab-2) state that 50% of the IGF-1 will be inhibited corresponding to the approximate level of inhibition observed in our assays.


The IGF-1/IGF-1R signaling axis in the skin: a new role for the dermis in aging-associated skin cancer.

Lewis DA, Travers JB, Somani AK, Spandau DF - Oncogene (2009)

IGF-1 secreted by normal human fibroblasts can protect keratinocytes from UVB-induced apoptosis(A) To determine if normal human fibroblasts can produce a protective factor which can activate the IGF-1R and restore the normal UVB response of keratinocytes, normal human fibroblasts were grown in the EpiLife NoIn media for 48 h (referred to as Conditioned Media, CM). Keratinocytes were grown in EpiLife NoIn media for 48 h and subsequently the media was replaced with EpiLife Complete media, EpiLife NoIn media, or EpiLife NoIn-CM derived from fibroblasts. Aliquots of EpiLife Complete media and CM-NoIn were treated with a neutralizing antibody to IGF-1 (Calbiochem, α-IGF-1, Ab2; inhibits 50% at suggested concentration) 15 min prior to adding it to the keratinocytes. One hour following media exchange, the keratinocytes were irradiated with 0 or 400 J/m2 of UVB. Six hours post-irradiation, the keratinocytes were harvested and assayed for the induction of apoptosis. Error bars indicate the standard error of the mean; the asterisk indicates significant difference between caspase 3 specific activities derived from keratinocytes grown in Complete media versus NoIn media (p<0.01, t-test). The data presented represent three independent assays. (B and C) Fibroblasts were treated with IGF-1-specific siRNA or non-specific siRNA for 24 h. NoIn media was then added to the fibroblasts for 48 h. At that time, the media was removed, filter-sterilized, and added to keratinocyte cultures. (B) qRT-PCR analysis of IGF-1 expression in fibroblasts following the indicated siRNA treatment. Error bars indicate the standard error of the mean; asterisk indicates significant (p<0.02, t-test) difference from nonsense siRNA-treated keratinocytes. (C) Keratinocytes were grown in NoIn media for 48 h, then the indicated media was added for 1 h. At that time, the keratinocytes were irradiated with the indicated dose of UVB and harvested six hours post-irradiation for caspase 3 analysis. Error bars indicate the standard error of the mean; the asterisks indicate significant difference between caspase 3 specific activities derived from keratinocytes grown in NoIn media + !GF-1 (p<0.03, t-test). The data presented represent three independent assays.
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Figure 2: IGF-1 secreted by normal human fibroblasts can protect keratinocytes from UVB-induced apoptosis(A) To determine if normal human fibroblasts can produce a protective factor which can activate the IGF-1R and restore the normal UVB response of keratinocytes, normal human fibroblasts were grown in the EpiLife NoIn media for 48 h (referred to as Conditioned Media, CM). Keratinocytes were grown in EpiLife NoIn media for 48 h and subsequently the media was replaced with EpiLife Complete media, EpiLife NoIn media, or EpiLife NoIn-CM derived from fibroblasts. Aliquots of EpiLife Complete media and CM-NoIn were treated with a neutralizing antibody to IGF-1 (Calbiochem, α-IGF-1, Ab2; inhibits 50% at suggested concentration) 15 min prior to adding it to the keratinocytes. One hour following media exchange, the keratinocytes were irradiated with 0 or 400 J/m2 of UVB. Six hours post-irradiation, the keratinocytes were harvested and assayed for the induction of apoptosis. Error bars indicate the standard error of the mean; the asterisk indicates significant difference between caspase 3 specific activities derived from keratinocytes grown in Complete media versus NoIn media (p<0.01, t-test). The data presented represent three independent assays. (B and C) Fibroblasts were treated with IGF-1-specific siRNA or non-specific siRNA for 24 h. NoIn media was then added to the fibroblasts for 48 h. At that time, the media was removed, filter-sterilized, and added to keratinocyte cultures. (B) qRT-PCR analysis of IGF-1 expression in fibroblasts following the indicated siRNA treatment. Error bars indicate the standard error of the mean; asterisk indicates significant (p<0.02, t-test) difference from nonsense siRNA-treated keratinocytes. (C) Keratinocytes were grown in NoIn media for 48 h, then the indicated media was added for 1 h. At that time, the keratinocytes were irradiated with the indicated dose of UVB and harvested six hours post-irradiation for caspase 3 analysis. Error bars indicate the standard error of the mean; the asterisks indicate significant difference between caspase 3 specific activities derived from keratinocytes grown in NoIn media + !GF-1 (p<0.03, t-test). The data presented represent three independent assays.
Mentions: Proper function of the skin requires the synergistic interactions between dermal fibroblasts and epidermal keratinocytes. The proliferation, differentiation, and stress responses of keratinocytes are greatly altered when deficiencies in dermal fibroblasts occur. We hypothesize that the normal UVB-response (defined as protection from carcinogenic initiation) of keratinocytes is also highly dependent on the status of dermal fibroblasts. As described above, we have defined conditions where the appropriate UVB-response of normal human keratinocytes grown in tissue culture can be altered by differential activation of the IGF-1R. We believe these culture conditions that alter the UVB-response in keratinocytes in vitro reflect instances of deficient fibroblast function in vivo. To determine if normal human fibroblasts can produce a protective factor which can activate the IGF-1R and restore the normal UVB response of keratinocytes, normal human fibroblasts were grown in the EpiLife NoIn media for forty-eight hours (referred to as Conditioned Media, NoIn-CM) and then the NoIn-CM was removed from the fibroblasts and filter sterilized. Keratinocytes were grown in EpiLife NoIn media for 48 hours and subsequently the media was replaced with EpiLife Complete media, with NoIn media, or NoIn-CM from fibroblasts. One hour following media exchange, the keratinocytes were irradiated with 0 or 400 J/m2 of UVB and subsequently assayed for the induction of apoptosis. As seen in Figure 2A, UVB irradiation induced a modest induction of apoptosis in keratinocytes treated with EpiLife Complete media and substantial apoptosis in keratinocytes grown in NoIn media. However, keratinocytes treated with NoIn-CM were resistant to UVB-induced apoptosis, similar to those treated with EpiLife Complete media. These data indicate that normal human fibroblasts produced a diffusible factor into the EpiLife insulin-deficient media that protected the keratinocytes from UVB-induced apoptosis. To determine if the protective factor produced by fibroblasts was IGF-1, we added a neutralizing antibody specific to IGF-1 to the fibroblast NoIn-CM. Addition of the IGF-1 neutralizing antibody prevented complete protection of UVB-induced apoptosis (Figure 2A). As expected, the addition of the neutralizing IGF-1 antibody to EpiLife Complete media did not alter the keratinocyte UVB response indicating a lack of cross-reactivity to the insulin in the EpiLife Complete media by the anti-IGF-1 antibody. At the concentration of IGF-1 antibody used, the manufacturers (Calbiochem, α-IGF-1 Ab-2) state that 50% of the IGF-1 will be inhibited corresponding to the approximate level of inhibition observed in our assays.

Bottom Line: In human skin, epidermal keratinocytes do not express IGF-1, and hence the IGF-1 receptor on keratinocytes is activated by IGF-1 secreted from dermal fibroblasts.Finally, the appropriate UVB response is restored in geriatric skin in vivo through pretreatment with exogenous IGF-1.These studies provide further evidence for a role of the IGF-1 receptor (IGF-1R) in suppressing UVB-induced carcinogenesis, suggest that fibroblasts have a critical role in maintaining appropriate activation of the keratinocyte IGF-1R, and imply that reduced expression of IGF-1 in geriatric skin could be an important component in the development of aging-related non-melanoma skin cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.

ABSTRACT
The appropriate response of human keratinocytes to ultraviolet-B (UVB) is dependent on the activation status of the insulin-like growth factor 1 (IGF-1) receptor. Keratinocytes grown in conditions in which the IGF-1 receptor is inactive inappropriately replicate in the presence of UVB-induced DNA damage. In human skin, epidermal keratinocytes do not express IGF-1, and hence the IGF-1 receptor on keratinocytes is activated by IGF-1 secreted from dermal fibroblasts. We now show that the IGF-1 produced by human fibroblasts is essential for the appropriate UVB response of keratinocytes. Furthermore, the expression of IGF-1 is silenced in senescent fibroblasts in vitro. Using quantitative reverse transcriptase-PCR and immunohistochemisty, we can show that IGF-1 expression is also silenced in geriatric dermis in vivo. The diminished IGF-1 expression in geriatric skin correlates with an inappropriate UVB response in geriatric volunteers. Finally, the appropriate UVB response is restored in geriatric skin in vivo through pretreatment with exogenous IGF-1. These studies provide further evidence for a role of the IGF-1 receptor (IGF-1R) in suppressing UVB-induced carcinogenesis, suggest that fibroblasts have a critical role in maintaining appropriate activation of the keratinocyte IGF-1R, and imply that reduced expression of IGF-1 in geriatric skin could be an important component in the development of aging-related non-melanoma skin cancer.

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Related in: MedlinePlus