Serum starvation induces density-dependent apoptosis via HIF-1 activation and JNK suppression

Abstract

Serum deprivation is a well-established inducer of apoptosis, yet the molecular mechanisms governing this process remain incompletely understood. Here, we show that serum starvation selectively triggers intrinsic apoptosis in high-density murine embryonic fibroblasts (MEFs) through coordinated HIF-1α activation and JNK signaling suppression. Knockdown of HIF-1α abolishes caspase-3 activation and apoptosis induced by serum deprivation, whereas upregulation of HIF-1α in low-density cells recapitulates the apoptotic response observed in high-density cultures. Simultaneously, serum deprivation leads to the suppression of the JNK pathway, which contributes to apoptosis. Notably, combined HIF-1α activation and JNK inhibition in low-density cells fully mimics the apoptotic phenotype of high-density conditions, underscoring the interplay between these pathways. Together, these findings define a density-dependent apoptotic switch in which HIF-1α drives metabolic stress adaptation, whereas JNK suppression removes a critical survival signal, converging to promote mitochondrial-mediated cell death. This work provides a mechanistic framework for understanding nutrient stress-induced apoptosis and suggests potential therapeutic targets for diseases characterized by aberrant cell survival.

1. Introduction

Apoptosis, a genetically programmed form of cell death, plays pivotal roles in maintaining tissue homeostasis and eliminating damaged or unnecessary cells during development and disease [1]. Unlike necrosis, apoptosis is characterized by distinct morphological changes, including cell shrinkage, chromatin condensation, and the formation of apoptotic bodies, which are efficiently cleared by phagocytes without inducing inflammation [2]. This process is orchestrated by conserved molecular machinery involving initiator caspases (e.g., caspase-8 and -9), executioner caspases (e.g., caspase-3 and -7), and regulatory proteins such as the Bcl-2 family and inhibitor of apoptosis proteins (IAPs) [3,4]. Dysregulation of apoptotic pathways contributes to diverse pathologies, including cancer, neurodegeneration, and autoimmune disorders [5].

Two principal pathways govern apoptosis: the extrinsic pathway, which is initiated by death receptor activation (e.g., Fas and TNF-R1), and the intrinsic pathway, which is triggered by mitochondrial outer membrane permeabilization (MOMP) [6]. The extrinsic pathway recruits caspase-8 via the death-inducing signaling complex (DISC), whereas the intrinsic pathway involves cytochrome c release and apoptosome-mediated caspase-9 activation [7,8]. Cross-talk between these pathways occurs through Bid cleavage, which links caspase-8 activation to mitochondrial amplification [9].

Serum starvation, a well-established model for studying apoptosis induced by trophic factor deprivation, triggers cell death pathways depending on the cellular context and microenvironmental conditions [10–12]. The classic intrinsic (mitochondrial) apoptotic pathway has dominated mechanistic studies of serum starvation-induced apoptosis [13]. While serum withdrawal broadly disrupts survival signaling by removing growth factors and nutrients, emerging evidence suggests that cell density critically modulates the molecular mechanisms of apoptosis execution [14,15]. However, the precise regulatory networks governing this density-dependent cell death or cell survival are still unclear.