Project description:To understand the impact of dysfunctional TRPV3 on keratinocyte proliferation and differentiation, we conducted transcriptomic analysis on cells that express cloned different TRPV3 mutations identified in focal palmoplantar keratoderma (FPPK) patients and identified a number of perturbed pathways associated with epidermal cells construction and development. We also analyzed the molecular changes of skin biopsy samples from patients. Our data suggests that TRPV3 dysfunction inhibits keratinocyte differentiation, actives apoptosis pathway resulted in cell death, and disturbs the balance between keratinocyte proliferation and differentiation processes in the skin. To explore the functional impact of the different TRPV3 mutations on cells, we cloned the wild type and mutant TRPV3 clones into expression vector. The wild-type human TRPV3 gene was amplified from cDNA made from a mixture of human tissue total RNA and cloned into pcDNA3.1 (Life Technologies, Carlsbad, CA). The accuracy of the cloned sequence was checked with Sanger sequencing. Quick Change II XL site-directed mutagenesis kit (Agilent, Santa Clara, CA) was used to introduce sequence changes corresponding to mutations observed in patients. The HEK293T cells were transfected using Lipofectamine 2000 (Life Technologies, Carlsbad, CA) according to the manufacturerâs instructions. Cells were also transfected with the same plasmid without insert as control.
Project description:To understand the impact of dysfunctional TRPV3 on keratinocyte proliferation and differentiation, we conducted transcriptomic analysis on cells that express cloned different TRPV3 mutations identified in focal palmoplantar keratoderma (FPPK) patients and identified a number of perturbed pathways associated with epidermal cells construction and development. We also analyzed the molecular changes of skin biopsy samples from patients. Our data suggests that TRPV3 dysfunction inhibits keratinocyte differentiation, actives apoptosis pathway resulted in cell death, and disturbs the balance between keratinocyte proliferation and differentiation processes in the skin.
Project description:We identified a number of affected pathways through transcriptome analysis on the skin biopsy samples of the FPPK patients. Our findings suggest that TRPV3 dysfunction may increase apoptotic activity, inhibit keratinocyte differentiation and disturb the intricate balance between proliferation and differentiation state of keratinocytes in the skin. To understand the effect of TRPV3 mutation, transcriptome of HaCaT cell lines transfected with mutant TRPV3 were profiled in time-course manner (16, 24 and 40hr).
Project description:We identified a number of affected pathways through transcriptome analysis on the skin biopsy samples of the FPPK patients. Our findings suggest that TRPV3 dysfunction may increase apoptotic activity, inhibit keratinocyte differentiation and disturb the intricate balance between proliferation and differentiation state of keratinocytes in the skin. To understand the effect of TRPV3 mutation, transcriptome of 293T cell lines transfected with mutant TRPV3 were profiled in time-course manner (16, 24 and 40hr).
Project description:Palmoplantar skin is structurally and functionally unique, but the transcriptional programs driving this specialization are unknown. Here, we exploit bulk and single-cell RNA-sequencing of human palm, sole, and hip skin to describe the distinguishing characteristics of palmoplantar and non-palmoplantar skin while also uncovering previously unappreciated differences between palmar and plantar sites. Our approach reveals downregulation of diverse immunological processes and decreased immune cell populations in palmoplantar skin, highlighting an altered immune environment in the skin of the palms and soles. Further, we identify specific palmoplantar and non-palmoplantar fibroblast populations that appear to orchestrate key differences in cell-cell communication in palm, sole, and hip. Dedicated analysis of epidermal keratinocytes highlights major differences in basal cell fraction among the three sites and validates the presence of a more differentiated, cycling basal population. Finally, our data demonstrate the existence of two spinous keratinocyte populations that constitute two parallel, site-selective epidermal differentiation trajectories. Together, these results provide a deep characterization of the highly adapted palmoplantar skin and contribute new insights into the fundamental biology of human skin.
Project description:Palmoplantar skin is structurally and functionally unique, but the transcriptional programs driving this specialization are unknown. Here, we exploit single-cell RNA-sequencing of human palm, sole, and hip skin to describe the distinguishing characteristics of palmoplantar and non-palmoplantar skin while also uncovering previously unappreciated differences between palmar and plantar sites. Our approach reveals downregulation of diverse immunological processes and decreased immune cell populations in palmoplantar skin, highlighting an altered immune environment in the skin of the palms and soles. Further, we identify specific palmoplantar and non-palmoplantar fibroblast populations that appear to orchestrate key differences in cell-cell communication in palm, sole, and hip. Dedicated analysis of epidermal keratinocytes highlights major differences in basal cell fraction among the three sites and validates the presence of a more differentiated, cycling basal population. Finally, our data demonstrate the existence of two spinous keratinocyte populations that constitute two parallel, site-selective epidermal differentiation trajectories. Together, these results provide a deep characterization of the highly adapted palmoplantar skin and contribute new insights into the fundamental biology of human skin.
Project description:The goal of our study was to determine the effect of overexpressing TRPV3 in the lung epithelial cell line BEAS-2Bs. BEAS-2B cells, were transfected with hTRPV3 in pcDNA3.1V5/His and stable overexperssion was achieved using antibiotic selection (G418). Comparisons were between treatments in wild-type cells vs those overexperssing TRPV3.