PAPP-A-Specific IGFBP-4 Proteolysis in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

The insulin-like growth factors IGF-I and IGF-II-as well as their binding proteins (IGFBPs), which regulate their bioavailability-are involved in many pathological and physiological processes in cardiac tissue. Pregnancy-associated plasma protein A (PAPP-A) is a metalloprotease that preferentially cleaves IGFBP-4, releasing IGF and activating its biological activity. Previous studies have shown that PAPP-A-specific IGFBP-4 proteolysis is involved in the pathogenesis of cardiovascular diseases, such as ischemia, heart failure, and acute coronary syndrome. However, it remains unclear whether PAPP-A-specific IGFBP-4 proteolysis participates in human normal cardiomyocytes. Here, we report PAPP-A-specific IGFBP-4 proteolysis occurring in human cardiomyocytes derived from two independent induced pluripotent cell lines (hiPSC-CMs), detected both on the cell surface and in the cell secretome. PAPP-A was measured by fluoroimmune analysis (FIA) in a conditioned medium of hiPSC-CMs and was detected in concentrations of up to 4.3 ± 1.33 ng/mL and 3.8 ± 1.1 ng/mL. The level of PAPP-A-specific IGFBP-4 proteolysis was determined as the concentration of NT-IGFBP-4 proteolytic fragments using FIA for a proteolytic neo-epitope-specific assay. We showed that PAPP-A-specific IGFBP-4 proteolysis is IGF-dependent and inhibited by EDTA and 1,10-phenanthroline. Therefore, it may be concluded that PAPP-A-specific IGFBP-4 proteolysis functions in human normal cardiomyocytes, and hiPSC-CMs contain membrane-bound and secreted forms of proteolytically active PAPP-A.

IGF-Binding Proteins and Their Proteolysis as a Mechanism of Regulated IGF Release in the Nervous Tissue.

Insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) play a key role in the maintenance of the nervous tissue viability. IGF-1 and IGF-2 exhibit the neuroprotective effects by stimulating migration and proliferation of nervous cells, activating cellular metabolism, inducing regeneration of damaged cells, and regulating various stages of prenatal and postnatal development of the nervous system. The availability of IGFs for the cells is controlled via their interaction with the IGF-binding proteins (IGFBPs) that inhibit their activity. On the contrary, the cleavage of IGFBPs by specific proteases leads to the IGF release and activation of its cellular effects. The viability of neurons in the nervous tissue is controlled by a complex system of trophic factors secreted by auxiliary glial cells. The main source of IGF for the neurons are astrocytes. IGFs can accumulate as an extracellular free ligand near the neuronal membranes as a result of proteolytic degradation of IGFBPs by proteases secreted by astrocytes. This mechanism promotes interaction of IGFs with their genuine receptors and triggers intracellular signaling cascades. Therefore, the release of IGF by proteolytic cleavage of IGFBPs is an important mechanism of neuronal protection. This review summarizes the published data on the role of IGFs and IGFBPs as the key players in the neuroprotective regulation with a special focus on the specific proteolysis of IGFBPs as a mechanism for the regulation of IGF bioavailability and viability of neurons.

IGFBP-4 Proteolysis by PAPP-A in a Primary Culture of Rat Neonatal Cardiomyocytes under Normal and Hypertrophic Conditions.

Cardiovascular diseases (CVD) are among the leading causes of death and disability worldwide. Pregnancy-associated plasma protein-A (PAPP-A) is a matrix metalloprotease localized on the cell surface. One of the substrates that PAPP-A cleaves is the insulin-like growth factor binding protein-4 (IGFBP-4), a member of the family of proteins that bind insulin-like growth factor (IGF). Proteolysis of IGFBP-4 by PAPP-A occurs at a specific site resulting in formation of two proteolytic fragments - N-terminal IGFBP-4 (NT-IGFBP-4) and C-terminal IGFBP-4 (CT-IGFBP-4), and leads to the release of IGF activating various cellular processes including migration, proliferation, and cell growth. Increased levels of the proteolytic IGFBP-4 fragments correlate with the development of CVD complications and increased risk of death in patients with the coronary heart disease, acute coronary syndrome, and heart failure. However, there is no direct evidence that PAPP-A specifically cleaves IGFBP-4 in the cardiac tissue under normal and pathological conditions. In the present study, using a primary culture of rat neonatal cardiomyocytes as a model, we have demonstrated that: 1) proteolysis of IGFBP-4 by PAPP-A occurs in the conditioned medium of cardiomyocytes, 2) PAPP-A-specific IGFBP-4 proteolysis is increased when cardiomyocytes are transformed to a hypertrophic state. Thus, it can be assumed that the enhancement of IGFBP-4 cleavage by PAPP-A and hypertrophic changes in cardiomyocytes accompanying CVD are interrelated, and PAPP-A appears to be one of the activators of the IGF-dependent processes in normal and hypertrophic-state cardiomyocytes.