Trafficking of cholesterol from lipid droplets to mitochondria in bovine luteal cells: Acute control of progesterone synthesis

Abstract

The corpus luteum is a transient endocrine gland that synthesizes and secretes the steroid hormone, progesterone, which is vital for establishment and maintenance of pregnancy. Luteinizing hormone (LH) via activation of protein kinase A (PKA) acutely stimulates luteal progesterone synthesis via a complex process, converting cholesterol via a series of enzymatic reactions, into progesterone. Lipid droplets in steroidogenic luteal cells store cholesterol in the form of cholesterol esters, which are postulated to provide substrate for steroidogenesis. Early enzymatic studies showed that hormone sensitive lipase (HSL) hydrolyzes luteal cholesterol esters. In this study, we tested whether HSL is a critical mediator of the acute actions of LH on luteal progesterone production. Using LH-responsive bovine small luteal cells our results reveal that LH, forskolin, and 8-Br cAMP-induced PKA-dependent phosphorylation of HSL at Ser563 and Ser660, events known to promote HSL activity. Small molecule inhibition of HSL activity and siRNA-mediated knock down of HSL abrogated LH-induced progesterone production. Moreover, western blotting and confocal microscopy revealed that LH stimulates phosphorylation and translocation of HSL to lipid droplets. Furthermore, LH increased trafficking of cholesterol from the lipid droplets to the mitochondria, which was dependent on both PKA and HSL activation. Taken together, these findings identify a PKA/HSL signaling pathway in luteal cells in response to LH and demonstrate the dynamic relationship between PKA, HSL, and lipid droplets in luteal progesterone synthesis.

Luteinizing Hormone Regulation of Inter-Organelle Communication and Fate of the Corpus Luteum

“…It plays an essential role in lipid metabolism and is responsible for mediating the hydrolysis of di-and triacylglycerol, as well as cholesterol esters [160,161]. Hydrolysis of cholesterol esters by HSL in steroidogenic tissues provides substrate for the immediate synthesis of steroid hormones in adrenal glands, ovaries, and testis [58,[162][163][164]. One of the unique features of HSL that differentiates it from other lipases is that its activity against triacylglycerol and cholesteryl ester substrates is regulated by reversible phosphorylation [160].…”

“…Highly condensed chromatin is found at the nuclear periphery and in the central region of small luteal cells. However, in large luteal cells dispersed chromatin is found throughout the nucleus with condensed chromatin at the nuclear periphery [ 56 , 58 , 59 ]. The main functional differences between small and large luteal cells are that large luteal cells possess PGF2α receptor and produce 10- to 30-fold more progesterone than small luteal cells, whereas small luteal cells are highly responsive to treatment with LH and PKA activators [ 16 , 26 , 60 , 61 ].…”

“…In adipocytes, the translocation of HSL to the lipid droplets occurs by virtue of PLIN1 localization to the surface of lipid droplets, physically interacting with HSL within the N -terminal region of PLIN1 to initiate the hydrolysis of cholesterol esters. This mechanism is not prominent in bovine luteal cells that express little PLIN1 [ 58 , 59 ]. Exactly how HSL associates with luteal lipid droplets is presently unknown.…”

“…The notion that lipid droplets and mitochondria interact is well-supported. Luteal mitochondria also interact with lipid droplets at specific contact sites [ 58 , 59 ]. Proteomic analysis suggests that the luteal lipid droplets also interact with other key intracellular organelles involved in steroidogenesis and energy homeostasis, such as the ER, endosomes, and lysosomes [ 105 ].…”

“…AMPK regulates diverse metabolic events including inhibition of lipolysis and synthesis of fatty acids by phosphorylating HSL and acetyl-coA carboxylase alpha (ACACA) [ 184 ]. HSL interacts with lipid droplets and mediates the hydrolysis of cholesterol esters providing cholesterol substrate for immediate synthesis of steroid hormones [ 58 , 187 ]. ACACA catalyzes the carboxylation of acetyl-CoA to malonyl-CoA and is the rate-limiting enzyme in fatty acid synthesis.…”