Publications by authors named "Konduru Seetharama Sastry"

Melanocyte survival is mediated by diverse signaling pathways. However, the molecular mechanisms they use and molecules that they target are incompletely understood. Here, we show that melanocyte survival is mediated by diverse, nonredundant signaling pathways, including ERK1/2, AKT, PKA, and PKC. Each of these pathways is exerting prosurvival effects by phosphorylating the BAD. While Ser112-BAD phosphorylation is regulated by pERK, pPKA and pPKC, Ser136 and Ser155 phosphorylation are exclusively controlled by pAKT and pPKA, respectively. Inhibition of these pathways individually resulted in only modest apoptosis; however, most significant apoptosis, as a result of BAD dephosphorylation, was seen when all pathways were inhibited concurrently. BAD phosphorylation was essential for survival of melanocytes as cells expressing phosphorylation-deficient BAD were not rescued by any of the identified pathway. Furthermore, melanocytes became insensitive to kinase inhibitor-induced apoptosis when BAD expression was knocked down by BAD-shRNA. Overexpression of BAD in melanocytes stimulated faster apoptosis in response to kinase inhibitors. Taken together, our results show that BAD is acting as a convergence point for diverse survival pathways in melanocytes. Understanding the molecular mechanisms of melanocyte survival provides fundamental new insights into physiological mechanisms involved in the development of various melanocyte pathologies such as melanoma and vitiligo.

Oxidative stress is known to induce melanocyte death, but the underlying mechanisms are incompletely understood. To identify oxidative stress-induced global gene expression changes in melanocytes, we treated PIG1 melanocytes with HO in a dose- and time-dependent manner and performed RNA-seq. This approach allowed us to capture the events occurring early as well as late phase after treatment with HO. Our bioinformatics analysis identified differentially expressed genes involved in various biological processes of melanocytes which are known to contribute to the vitiligo development, such as apoptosis, autophagy, cell cycle regulation, cell adhesion, immune and inflammatory responses, melanocyte pluripotency, and developmental signaling such as WNT and NOTCH pathways. We uncovered several novel genes that are not previously described to be involved in melanocytic response to stress nor in vitiligo pathogenesis. Quantitative PCR and western blot analysis of selected proteins, performed on PIG1 and primary human epidermal melanocytes, confirmed the RNA-seq data. Interestingly, we discovered an aberrant regulation of several transcription factors that are involved in diabetes, neurological, and psychiatric diseases, all of which are comorbid conditions in patients with vitiligo. Our results may lead to a better understanding of the molecular mechanisms underlying vitiligo pathogenesis and help developing new drug targets for effective treatment.

Background: Functional brown adipose tissue (BAT), involved in energy expenditure, has recently been detected in substantial amounts in adults. Formerly overlooked BAT has now become an attractive anti-obesity target.

Methods And Results: Molecular characterization of human brown and white adipocytes, using a myriad of techniques including high-throughput RNA sequencing and functional assays, showed that PAZ6 and SW872 cells exhibit classical molecular and phenotypic markers of brown and white adipocytes, respectively. However, the pre-adipocyte cell line SGBS presents a versatile phenotype. A transit expression of classical brown markers such as UCP1 and PPARγ peaked and declined at day 28 post-differentiation initiation. Conversely, white adipocyte markers, including Tcf21, showed reciprocal behavior. Interestingly, leptin levels peaked at day 28 whereas the highest adiponectin mRNA levels were detected at day 14 of differentiation. Phenotypic analysis of the abundance and shape of lipid droplets were consistent with the molecular patterns. Accordingly, the oxidative capacity of SGBS adipocytes peaked on differentiation day 14 and declined progressively towards differentiation day 28.

Conclusions: Our studies have unveiled a new phenotype of human adipocytes, providing a tool to identify molecular gene expression patterns and pathways involved in the conversion between white and brown adipocytes.