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Decreased expression of KGF/FGF7 and its receptor in pathological hypopigmentation.

Purpura V, Persechino F, Belleudi F, Scrofani C, Raffa S, Persechino S, Torrisi MR - J. Cell. Mol. Med. (2014)

View Article: PubMed Central - PubMed

Affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Medicina Clinica e Molecolare, Sapienza Universita' di Roma, Roma, Italy.

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To the Editor: The molecular mechanisms and cellular pathways involved in cutaneous pigmentation, as well as thecrucial role played by the epidermal keratinocytes in the process, are just starting to beelucidated... In fact, a number of recent studies from different authors including our group havepointed out that the uptake by keratinocytes of the melanosomes released by the melanocytes occursthrough phagocytic ingestion and is regulated by the activity of some receptors, such asprotease-activated receptor-2 (PAR-2) and keratinocyte growth factor receptor/fibroblast growthfactor receptor 2b (KGFR/FGFR2b), followed by actin cytoskeleton reorganization... Dermalfibroblasts are known to participate in this complex cellular interplay controlling pigmentationthrough the modulated secretion of growth factors, some of them acting directly on the melanocytes and stimulating themelanogenesis, such as stem cell factor and basic fibroblast growth factor, while others promoting the melanosome phagocytic uptakeby the keratinocytes, as occurring in the case of keratinocyte growth factor/fibroblast growthfactor 7 (KGF/FGF7): in this context, in fact, we have proposed that the paracrine growth factorKGF, released from dermal fibroblasts, promotes melanosome transfer through binding to andactivation of its tyrosine kinase receptor KGFR, expressed on the keratinocytes, but not onmelanocytes or fibroblasts: the receptor signalling recruits and activates phospholipase Cγ,an essential player of the phagocytic process... Given thecrucial role of the secreted KGF/FGF7 in the modulation of the melanosome uptake by keratinocytes and taking advantage of our in vitromodels of melanosome transfer, we firstinvestigated here the efficiency of melanosome transfer in the above-mentioned hypopigmentationconditions as well as the ability of supernatants (SNs) collected from primary cultured human dermalfibroblasts, derived from the different lesional skin samples or from healthy donors as described inthe Data S1, to stimulate the process... To evaluate if the effects of the various SNs would be ascribed, at least in part, to thepresence of KGFR/FGFR2b ligands released in the fibroblast culture medium, as previouslydemonstrated in previous papers from our group, addition of the specific FGFR2tyrosine kinase inhibitor SU5402 was also performed: significant inhibition of the melanosome uptakewas found only when the inhibitor was added to the SN from NHFs or to the KGF-treated cultures (Fig. 1A, lower panels), suggesting a possible deficiency ofparacrine KGFR ligands in the pathological lesions... Then, to assess if the reduction of melanosometransfer in response to SNs from lesional fibroblasts would be dependent on an altered expression ofKGF, the growth factor mRNA transcript levels were analysed by real-time RT-PCR and normalized withrespect to β-actin, showing a clear decrease of KGF mRNA expression in all groups of HFsderived from lesional skin compared with the control NHFs (Fig. 1B)... In addition, ELISA test demonstrated that KGF protein levels were significantlydecreased in SNs from all lesional HFs compared with NHFs (Fig. 1C)... Interestingly, consistent with the mRNA expression data, the KGF released by vitiligoHFs was significantly reduced if compared with that secreted by both ND HFs and rSutton HFs (Fig. 1C)... Again, the additionof SU5402 was able to abolish the KGF effect in both cocultures (Fig. 2A, lower panels), providing a further evidence of the involvement of KGFRactivation and signalling in the process and suggesting a decreased receptor expression in thepathological condition... Therefore, with the aim to analyse the receptor expression, we quantifiedKGFR transcript levels by real-time RT-PCR and we found a decreased receptor mRNA expression in NDHKs compared with NHK control cells (Fig. 2C)... Thus, atleast in the ND disorder, low levels of KGFR might significantly contribute to the reduction ofKGF-mediated melanosome transfer... Taken together, our results further support the key roles played, on the melanosome transfer innormal skin, by KGF/FGF7 released by dermal fibroblasts and by its receptor KGFR/FGFR2b expressedand activated on the epidermal keratinocytes (Fig. 2D,cartoon on the left) and suggest a deficient expression of both players (Fig. 2D, cartoon on the right) as an additional pathogenic mechanism involved inhypopigmentary disorders.

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Decreased melanosome uptake ability and KGFR expression in keratinocytes from ND lesion.(A and B) Cocultures of MST-L melanoma cells with normal humankeratinocytes (NHKs) or with keratinocytes derived from the ND lesion (ND HKs) were treated withKGF. Immunofluorescence (A and B) and phase-contrast (B)images show that the tyrosinase-positive dots in ND HKs upon KGF treatment are strongly reduced withrespect to those in NHKs (A and B, circles) and that the addition ofSU5402 abolishes the KGF effect; bars: 10 μm. (C) Real-time RT-PCR reveals adecreased KGFR mRNA expression in ND HKs compared with NHK control cells. (D) Schematicdrawing showing the effects of decreased levels of KGF and KGFR on melanosome transfer inhypopigmented lesions.
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fig02: Decreased melanosome uptake ability and KGFR expression in keratinocytes from ND lesion.(A and B) Cocultures of MST-L melanoma cells with normal humankeratinocytes (NHKs) or with keratinocytes derived from the ND lesion (ND HKs) were treated withKGF. Immunofluorescence (A and B) and phase-contrast (B)images show that the tyrosinase-positive dots in ND HKs upon KGF treatment are strongly reduced withrespect to those in NHKs (A and B, circles) and that the addition ofSU5402 abolishes the KGF effect; bars: 10 μm. (C) Real-time RT-PCR reveals adecreased KGFR mRNA expression in ND HKs compared with NHK control cells. (D) Schematicdrawing showing the effects of decreased levels of KGF and KGFR on melanosome transfer inhypopigmented lesions.

Mentions: To evaluate the contribution of the lesional keratinocytes on the inefficient melanosometransfer, we focused our attention on the above ND biopsy, because of the postulated defect of theorganelle uptake in such disorder [11,12]. To dissect in vitro the process, wecocultured the MST-L melanocytes with primary keratinocytes derived from the ND (ND HKs) or fromnormal skin, at a seeding ratio of 1:40. Serum starvation and treatment with KGF in the presence orabsence of SU5402 were performed as above. The quantitative double immunofluorescence revealed thatthe KGF-induced increase of the tyrosinase-positive dots in the cytoplasm of ND HKs was much lowercompared with NHKs (Fig. 2A, middle panels). Brightfield andphase-contrast microscopy were used to unequivocally demonstrate the decreased melanosome transferto the lesional keratinocytes (Fig. 2B). Again, the additionof SU5402 was able to abolish the KGF effect in both cocultures (Fig. 2A, lower panels), providing a further evidence of the involvement of KGFRactivation and signalling in the process and suggesting a decreased receptor expression in thepathological condition. Therefore, with the aim to analyse the receptor expression, we quantifiedKGFR transcript levels by real-time RT-PCR and we found a decreased receptor mRNA expression in NDHKs compared with NHK control cells (Fig. 2C). Thus, atleast in the ND disorder, low levels of KGFR might significantly contribute to the reduction ofKGF-mediated melanosome transfer.


Decreased expression of KGF/FGF7 and its receptor in pathological hypopigmentation.

Purpura V, Persechino F, Belleudi F, Scrofani C, Raffa S, Persechino S, Torrisi MR - J. Cell. Mol. Med. (2014)

Decreased melanosome uptake ability and KGFR expression in keratinocytes from ND lesion.(A and B) Cocultures of MST-L melanoma cells with normal humankeratinocytes (NHKs) or with keratinocytes derived from the ND lesion (ND HKs) were treated withKGF. Immunofluorescence (A and B) and phase-contrast (B)images show that the tyrosinase-positive dots in ND HKs upon KGF treatment are strongly reduced withrespect to those in NHKs (A and B, circles) and that the addition ofSU5402 abolishes the KGF effect; bars: 10 μm. (C) Real-time RT-PCR reveals adecreased KGFR mRNA expression in ND HKs compared with NHK control cells. (D) Schematicdrawing showing the effects of decreased levels of KGF and KGFR on melanosome transfer inhypopigmented lesions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4302659&req=5

fig02: Decreased melanosome uptake ability and KGFR expression in keratinocytes from ND lesion.(A and B) Cocultures of MST-L melanoma cells with normal humankeratinocytes (NHKs) or with keratinocytes derived from the ND lesion (ND HKs) were treated withKGF. Immunofluorescence (A and B) and phase-contrast (B)images show that the tyrosinase-positive dots in ND HKs upon KGF treatment are strongly reduced withrespect to those in NHKs (A and B, circles) and that the addition ofSU5402 abolishes the KGF effect; bars: 10 μm. (C) Real-time RT-PCR reveals adecreased KGFR mRNA expression in ND HKs compared with NHK control cells. (D) Schematicdrawing showing the effects of decreased levels of KGF and KGFR on melanosome transfer inhypopigmented lesions.
Mentions: To evaluate the contribution of the lesional keratinocytes on the inefficient melanosometransfer, we focused our attention on the above ND biopsy, because of the postulated defect of theorganelle uptake in such disorder [11,12]. To dissect in vitro the process, wecocultured the MST-L melanocytes with primary keratinocytes derived from the ND (ND HKs) or fromnormal skin, at a seeding ratio of 1:40. Serum starvation and treatment with KGF in the presence orabsence of SU5402 were performed as above. The quantitative double immunofluorescence revealed thatthe KGF-induced increase of the tyrosinase-positive dots in the cytoplasm of ND HKs was much lowercompared with NHKs (Fig. 2A, middle panels). Brightfield andphase-contrast microscopy were used to unequivocally demonstrate the decreased melanosome transferto the lesional keratinocytes (Fig. 2B). Again, the additionof SU5402 was able to abolish the KGF effect in both cocultures (Fig. 2A, lower panels), providing a further evidence of the involvement of KGFRactivation and signalling in the process and suggesting a decreased receptor expression in thepathological condition. Therefore, with the aim to analyse the receptor expression, we quantifiedKGFR transcript levels by real-time RT-PCR and we found a decreased receptor mRNA expression in NDHKs compared with NHK control cells (Fig. 2C). Thus, atleast in the ND disorder, low levels of KGFR might significantly contribute to the reduction ofKGF-mediated melanosome transfer.

View Article: PubMed Central - PubMed

Affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Medicina Clinica e Molecolare, Sapienza Universita' di Roma, Roma, Italy.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

To the Editor: The molecular mechanisms and cellular pathways involved in cutaneous pigmentation, as well as thecrucial role played by the epidermal keratinocytes in the process, are just starting to beelucidated... In fact, a number of recent studies from different authors including our group havepointed out that the uptake by keratinocytes of the melanosomes released by the melanocytes occursthrough phagocytic ingestion and is regulated by the activity of some receptors, such asprotease-activated receptor-2 (PAR-2) and keratinocyte growth factor receptor/fibroblast growthfactor receptor 2b (KGFR/FGFR2b), followed by actin cytoskeleton reorganization... Dermalfibroblasts are known to participate in this complex cellular interplay controlling pigmentationthrough the modulated secretion of growth factors, some of them acting directly on the melanocytes and stimulating themelanogenesis, such as stem cell factor and basic fibroblast growth factor, while others promoting the melanosome phagocytic uptakeby the keratinocytes, as occurring in the case of keratinocyte growth factor/fibroblast growthfactor 7 (KGF/FGF7): in this context, in fact, we have proposed that the paracrine growth factorKGF, released from dermal fibroblasts, promotes melanosome transfer through binding to andactivation of its tyrosine kinase receptor KGFR, expressed on the keratinocytes, but not onmelanocytes or fibroblasts: the receptor signalling recruits and activates phospholipase Cγ,an essential player of the phagocytic process... Given thecrucial role of the secreted KGF/FGF7 in the modulation of the melanosome uptake by keratinocytes and taking advantage of our in vitromodels of melanosome transfer, we firstinvestigated here the efficiency of melanosome transfer in the above-mentioned hypopigmentationconditions as well as the ability of supernatants (SNs) collected from primary cultured human dermalfibroblasts, derived from the different lesional skin samples or from healthy donors as described inthe Data S1, to stimulate the process... To evaluate if the effects of the various SNs would be ascribed, at least in part, to thepresence of KGFR/FGFR2b ligands released in the fibroblast culture medium, as previouslydemonstrated in previous papers from our group, addition of the specific FGFR2tyrosine kinase inhibitor SU5402 was also performed: significant inhibition of the melanosome uptakewas found only when the inhibitor was added to the SN from NHFs or to the KGF-treated cultures (Fig. 1A, lower panels), suggesting a possible deficiency ofparacrine KGFR ligands in the pathological lesions... Then, to assess if the reduction of melanosometransfer in response to SNs from lesional fibroblasts would be dependent on an altered expression ofKGF, the growth factor mRNA transcript levels were analysed by real-time RT-PCR and normalized withrespect to β-actin, showing a clear decrease of KGF mRNA expression in all groups of HFsderived from lesional skin compared with the control NHFs (Fig. 1B)... In addition, ELISA test demonstrated that KGF protein levels were significantlydecreased in SNs from all lesional HFs compared with NHFs (Fig. 1C)... Interestingly, consistent with the mRNA expression data, the KGF released by vitiligoHFs was significantly reduced if compared with that secreted by both ND HFs and rSutton HFs (Fig. 1C)... Again, the additionof SU5402 was able to abolish the KGF effect in both cocultures (Fig. 2A, lower panels), providing a further evidence of the involvement of KGFRactivation and signalling in the process and suggesting a decreased receptor expression in thepathological condition... Therefore, with the aim to analyse the receptor expression, we quantifiedKGFR transcript levels by real-time RT-PCR and we found a decreased receptor mRNA expression in NDHKs compared with NHK control cells (Fig. 2C)... Thus, atleast in the ND disorder, low levels of KGFR might significantly contribute to the reduction ofKGF-mediated melanosome transfer... Taken together, our results further support the key roles played, on the melanosome transfer innormal skin, by KGF/FGF7 released by dermal fibroblasts and by its receptor KGFR/FGFR2b expressedand activated on the epidermal keratinocytes (Fig. 2D,cartoon on the left) and suggest a deficient expression of both players (Fig. 2D, cartoon on the right) as an additional pathogenic mechanism involved inhypopigmentary disorders.

Show MeSH
Related in: MedlinePlus