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Page 581 Loh et al. Extracell Vesicles Circ Nucleic Acids 2023;4:568-87 https://dx.doi.org/10.20517/evcna.2023.34

PTHrP
PTHrP is a 36 amino acid neuroendocrine peptide and survival factor and is synthesized by proteolytic
cleavage from a prohormone, packaged into classical secretory vesicles (DCVs) and secreted via the
regulated secretory pathway [Figure 1]. PTHrP is secreted from parathyroid cells, adrenal medulla, pituitary,
pancreatic islets and the central nervous system, and in cancer cells. It is also secreted from osteoblastic cells
and keratinocytes via the constitutive pathway [Figure 1]. PTHrP acts as a hormone by binding to a
receptor, and some of its functions include stimulating adipocyte lipolysis , regulating calcium
[132]
[132]
homeostasis, and recruiting osteoblasts for bone formation . PTHrP is another example of a RSP protein
found in sEVs. sEVs derived from Lewis Lung carcinoma (LLC) containing PTHrP have been shown to
[110]
induce lipolysis in murine 3T3-L1 adipocytes . Condition medium from LLC cells exposed to Rab27A
shRNA to inhibit EV release had a lower effect on lipolysis than that from control cells, confirming that
LLC-EVs contribute to lipolysis. These LLC EVs fused directly with 3T3-L1 cells and were internalized in a
caveolin /raft-mediated manner, transferring PTHrP to the recipient cells to induce lipolysis. This lipolytic
activity was blocked by the presence of PTHrP neutralizing antibody. After centrifugation, the supernatant
fraction of medium containing PTHrP secreted from constitutive secretory vesicles from LLC cells or
recombinant PTHrP stimulated lipolysis when incubated with 3T3-L1 cells, indicating a dual mechanism of
PTHrP release from the LLC cells. Knocking down PTHrP receptor expression in 3T3-L1 cells eliminated
the lipolytic activity induced by LLC-derived sEVs, suggesting that the effect is dependent on extracellular
PTHrP receptor. Since this study has shown that the LLC-derived EVs containing PTHrP are fused with the
3T3-L1 cells and internalized, the mechanism by which the sEV cargo, PTHrP, is able to get recycled and
secreted to get exposure to the extracellular receptor to mediate its activity is unclear. Nevertheless, the data
presented do support the release of PTHrP by a dual mechanism via secretory vesicles and in sEVs. In this
case, PTHrP from the two secretory mechanisms performed a similar function. One can suggest that the
delivery of PTHrP in the circulation to target cells at a distance would be better protected from degradation
inside EVs.

Other RSP proteins trafficked to sEVs
With new proteomic data emerging for sEV, more RSP cargo proteins have been found to be associated
with these organelles. Among these is APP, which is synthesized in neurons and astrocytes. In neuron-like
chromaffin cells, APP is processed to β-amyloid peptides (Aβ1-40 and Aβ1-42), which are co-secreted with
peptide neurotransmitters (Galanin) and catecholamines from DCVs upon stimulation. APP, along with
secretases that convert APP into Aβ peptides, were present in purified DCVs. β-Amyloid peptides have also
been reported to be released from sEV [109,116] . Aβ1-42 levels in immunoaffinity-purified NDEVs from plasma
[116]
have been shown to be higher in AD patients compared to controls .

Another example is the granins which are trafficked into DCVs and released in a regulated manner [15,16] .
Some of these granins are processed into bioactive peptides. Proteomic analysis revealed CgA in the sEV of
human-induced pluripotent stem cell neurons expressing mutant presenilin 1. However, CgA was not in the
[113]
sEV of control cells . This suggests that CgA may be trafficked to sEV under certain pathological
conditions.

CONCLUDING REMARKS AND FUTURE DIRECTIONS
Here, we have summarized advancements in the molecular mechanisms for prohormone/neurotrophic
factor sorting, packaging, and transport in secretory vesicles for stimulated release from endocrine cells and
neurons. Recent studies have revealed that RSP proteins (examples above) are also trafficked to extracellular
vesicles/sEVs in neuroendocrine and cancer cells. Moreover, in one example, CPE/NF-α1, the physiological
functions may be different depending upon whether it is released from classical secretory vesicles (DCVs) or
in sEVs. It will not be surprising if RSP proteins within sEVs have different intracellular roles after they are