CHARACTERISTICS AND ROLES OF EXTRACELLULAR VESICLES RELEASED BY EPIDERMAL KERATINOCYTES

Key words: extracellular vesicles, epidermal keratinocytes. Manuscript word count: 3358 Table count: 2 Figure count: 3 Authors: Uyen Thi Trang Than1, David I Leavesley2, 3, Tony J Parker3, 4 Affiliation: 1Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec International Hospital, Ha Noi, Vietnam. 2Skin Research Institute of Singapore, Agency for Science, Technology and Research, Singapore. 3School of Biomedical Science, Faculty of Health, Queensland University of Technology, Brisbane, QLD, Australia. 4Tissue Repair and Translational Physiology Program, Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, QLD, Australia. Corresponding Author: Tony Parker, Institute of Health and Biomedical Innovation (IHBI), 60 Musk Avenue, Kelvin Grove, QLD, Australia, 4059. Email: a.parker@qut.edu.au. Tel: +61 7 3138 6187. Conflict of interest: There is no conflict of interest Abstract Keratinocytes, which constitute 90 % of the cells in the epidermis of the skin, have been demonstrated

to communicate with other skin cells such as fibroblasts, melanocytes and immune cells through

extracellular vesicles (EVs). This communication is facilitated by the enriched EV biomolecular cargo

which regulates multiple biological processes within skin tissue, including cell proliferation, cell

migration, anti-apoptosis, pigmentation transfer, and extracellular matrix (ECM) remodelling. This

review will provide an overview of the current literature and advances in the field of keratinocyte-derived

EV research with particular regard to the interactions and communication between keratinocytes and

other skin cells, mediated by EVs and EV components. Importantly, this information may shed some

light on the potential for keratinocyte-derived EVs in future biomedical studies.

Introduction

Keratinocytes are the predominant cell type in the epidermis of the skin, the largest and most superficial

organ of the body. The primary function of skin is to act as a barrier, protecting the internal organs from

external insults while maintaining a stable internal physiology. The development and function of skin

depends on many internal and external factors and thus the effects of communication between cells and

tissue is critical. Only recently, has it been appreciated that extracellular vesicles (EVs), secreted into

interstitial (i.e. extracellular) spaces, can mediate communication between keratinocytes and other cells

in the skin [1, 2]. This previously unknown mechanism of communication delivers functional molecules

between adjacent cells and alters cell behaviours such as proliferation and migration, or pigment

production [3, 4]. These recent novel findings have stimulated renewed interest in the underlying

physiological mechanisms that support the diverse and multiple functions performed by skin.

Extracellular vesicles are small membrane-enclosed vesicles released by the majority of cell types. EVs

can be classified into three different categories, based on their biogenesis. These include apoptotic bodies,

microvesicles, and exosomes which have size distributions which overlap [5]. Apoptotic bodies are the

largest in size ranging from 1 to 5 µm in diameter and are products of apoptosis [5]. Conversely,

microvesicles range in size from 100 to 1000 nm and are shed directly from cellular membrane through

a complex interplay between phospholipid redistribution and cytoskeletal rearrangement. Exosomes,

have the smallest diameter between 50 to 150 nm and are products of endocytosis and exocytosis [5].

After release, EVs have the capacity to transfer cargoes that are packaged during vesicle formation, to

recipient target cells, adding to their biochemical complement and potentially altering specific functions

of the recipient cell. Cargo delivery can be accomplished through one of three mechanisms: (i) specific

ligand / receptor interaction between integral proteins of EV and target cell membranes, (ii) direct

membrane fusion of EVs with plasma membrane of target cells, or (iii) endocytosis of EVs into the

cytoplasm of target cells [6]. In this manner, inter-cellular communication enables EVs to integrate and

coordinate biological processes within tissue, such as immune response, cell growth, differentiation,

organ formation, or tissue repair [6].

In this article we will discuss the biological role and sequelae of EVs released by epidermal keratinocytes.

It includes factors that stimulate the secretion EVs by keratinocytes, EV composition and cargoes.

Additionally, we will also discuss the intimate interactions that take place between keratinocytes through

EVs with other cells that comprise mammalian skin and contribute to the many functions provided by

skin.

Factors that stimulate secretion of extracellular vesicles from keratinocytes

It is widely recognised that EVs are secreted by most cell types into culture medium and body fluids [6,

7]. Moreover, some evidence suggests that EV composition and secretion may be subject to the specifics

of various pathological / stimulatory conditions [8]. For example, plasma exosomes from patients with

melanoma have higher levels of CD63 and CAV1 compared with healthy individuals indicating that the

specific composition of EVs may depend on the physiological state of parental cells [9]. Alternatively,

the EV composition may depend on the parental cell type, for instance, lung epithelial cells secrete EVs

containing lung surfactant proteins [10] while mature dendritic cells secrete exosomes enriched with

major histocompatibility complex (MHC) class II and intercellular adhesion molecule 1 (ICAM-1) [11].

Regarding keratinocytes, Bruth et al. first reported evidence for keratinocyte-derived EVs in 2004

describing them as HaCaT derived “plasma membrane vesicles” [12]. Since that initial report, several

investigators have studied keratinocyte-derived EV biogenesis and cargoes, which are influenced by

various stimulants and, in turn, determine their bioactivity.

Extracellular vesicle secretion is induced by a variety of exogenous stimuli [13]. When cells are exposed

to specific stimuli, EVs containing cargoes that reflect the secreting cell’s response to the stimulating

condition are actively exported. Currently available data characterising EV’s secreted by human

keratinocytes, reflect responses to serum-starvation (a.k.a. conditioned serum-free culture medium), EV

depleted serum, growth factors, Ca2+, irradiation, and hypoxia (Table 1) [1-3, 12, 14-19]. Regarding

serum, a poorly defined yet highly complex mixture of bioactive substances is commonly included in

culture media to support the survival and growth of mammalian cells [20], yet itself includes substantial

number of EVs of bovine origin. Serum starvation deprives cells of essential (largely ill-defined) growth

factors, hormones, attachment and survival factors required to support cell viability through phenotypic

and metabolic pathways [13, 21]. Under serum-free conditions with or without defined supplements

mammalian cells undergo severe stress that is communicated to adjacent cells by secreted EVs [21, 22].

An alternative approach to avoid stressing cells during EV studies is to deplete serum EVs by

centrifugation that has also been described as media for EV secretion [15, 23]. Unsurprisingly, almost

any stimuli, exogenous factor, or event that causes cellular stress results in increased export of EVs. In

addition to serum-free and EV-depleted serum medium, other stimuli known to induce keratinocytes to

release EVs include irradiation [18], poly (I:C) which is an immunostimulant and present in some viruses

[15], TGFα [1], and hypoxia [19]. Chemical reagents that lead to changes in cell physiology, for example

Ca2+ mobilisation (e.g. via monensin, or ionomycin), are reported to stimulate both keratinocyte

differentiation and the generation of gigantic multivesicular bodies and release of exosomes [17].

Importantly, undifferentiated and differentiated keratinocytes subjected to Ca2+ mobilisation export EVs

with distinct and unique cargo composition [17]. These findings may partially explain the corroboration

evident between physiological Ca2+ gradients evident in the epidermis and the increased expression of

particular proteins, for example 14-3-3 protein isoforms, in exosomes. Additionally, treating

keratinocytes in vitro with poly (I:C) and TGFα under serum-free conditions stimulates the secretion of

EVs specifically enriched with IL-36γ and HSP90α respectively [1, 15]. Other stressors, for example

irradiation and hypoxia, have been shown to produce EVs that when isolated and subsequently added to

naïve cells (i.e. not exposed to irradiation or hypoxia) stimulated the ‘bystander effect’, a phenomenon

where naïve cells express physiological responses of exposure to irradiation, or hypoxia; presumably as

a result of signals received from affected cells. When exposed to EVs collected from cells not exposed

to irradiation or hypoxia, naïve keratinocytes failed to express the ‘bystander effect’ [18, 19]. These data

represent the most compelling evidence among published data demonstrating the physiological state of

cells has a critical influence on the release of EVs, the composition of EV cargoes, and that EVs are

capable of promulgating cell-free paracrine communication. While some might argue that this is a niche

observation, it is not without wider implications for diseases and maladies with known but little

understood ‘off-target’ effects. Thus further investigation of which EV components could be used to

identify the hallmarks of particular cell types or diseases is warranted.

Keratinocyte-derived EV components

The evidence discussed above indicates that EV cargoes can have biological consequences. The binding

of EVs and release of cargoes into the recipient cell cytoplasm can cause significant physiological

consequences on target cells, altering cellular biochemistry and affecting cell functions and phenotype.

Consequently, this is an emerging focus of research and investigation. While relatively limited data is

available at the time of writing, several teams have reported data on both keratinocyte-derived EV

secretion and cargoes [14, 16, 17]. One aspect that is handicapping the field is that an agreed consensus

has yet to be reached for what constitutes a marker or markers for keratinocyte-derived EVs. Several

candidate species are popular in EVs released from all cell types, such as Alix, CD9, CD63, CD81,

TSG101, HSP70 [3, 15-17], however, not all are expressed in all EVs. Additional molecules, such as

metabolic enzymes, cytoskeletal proteins, signalling proteins, trafficking and adhesion proteins,

identified in EVs isolated from keratinocytes, may also be detected in EVs secreted by other cells [17,

24, 25]. Specific molecular cargoes are predicted to be associated with specific cell sources. Cytokeratins,

for example, are indicative of epithelia, and thus may be characteristic for keratinocyte-derived EVs [24].

However, it is now understood that the composition and abundance of specific candidate species can

depend on cell status and events that stimulate EV secretion. For instance, migrating keratinocytes

package cathepsin B into secreted EVs that communicate with other keratinocytes during wound healing

[12]. Similarly, some glycoprotein species which are important to the wound healing process, e.g.

fibroblast growth factor binding protein (FGF-BP), matrix metalloproteinase-1 (MMP-1), MMP-3/tissue

inhibitor of metalloproteinase-1 (MMP-3/TIMP-1) complex, MMP-8, MMP-9, plasminogen activator

inhibitor type-1 (PAI-1), transforming growth factor-β (TGF-β), and lactate dehydrogenase (LDH), have

been characterised in exosomes from undifferentiated and terminally differentiated keratinocytes [17].

Interestingly, seven 14-3-3 protein isoforms have been detected in exosomes released by keratinocytes,

however three isoforms (σ, ε, and τ) are only detectable in exosomes from differentiated keratinocytes

and not in exosomes from undifferentiated keratinocytes (Table 2) [17]. Furthermore, the abundance of

individual 14-3-3 protein isoforms were found to be distinct in exosomes isolated from either

differentiated or undifferentiated cell populations. For instance, 14-3-3 protein η is more abundant in

exosomes from undifferentiated keratinocytes than it is in exosomes from differentiated keratinocytes

[17]. In contrast, 14-3-3 protein β is expressed abundantly in exosomes from differentiated keratinocytes

compared to exosomes isolated from undifferentiated keratinocytes [17].

While proteins and glycoproteins are commonly utilised as EV molecular markers, a new class of robust

markers have recently emerged. MicroRNA species have been demonstrated to exhibit some specificity

to cell-type and pathology [26]. EV cargo inventories from sources as diverse as conditioned culture

media, plasma, saliva, and urine include multiple microRNA populations [7, 27-29]. Using next-

generation sequencing, we identified microRNAs in EVs isolated from human keratinocytes from

‘normal’ donor skin that were distinct from those detected in immortalised keratinocytes (HaCaT) [16].

We determined that the microRNAs let 7, hsa-miR 22, hsa-miR 27b and hsa-miR 21 were common to

all keratinocyte-derived EVs tested [16]. However, when the total microRNA profile identified in EVs

and their expression levels were subjected to Euclidean distance analysis, microRNA abundance in EVs

could discriminate between the following EV populations apoptotic bodies (1000 nm – 5000 nm),

microvesicles (100 nm – 1000 nm), and exosomes (30 nm –100 nm) [16]. We also found that the analysis

of the downstream target genes of these miRNA’s using bioinformatics is also effective at discriminating

exosomes, microvesicles and apoptotic bodies from each other [16]. Thus, we hypothesise that this is

evidence for the selective sorting of microRNA cargoes into exosomes. Of note, selective sorting of EV

cargoes also depends on the tissue origin of secreting cells [30-32].

Interaction of keratinocyte with other skin cells through EVs

Cell-to-cell communication, sending and receiving signals, is mediated by specific interactions between

molecular species in the extracellular environment, pericellular space of adjacent cells and specific

plasma membrane-associated molecules: ligands and receptors. Cell-to-cell communication is

fundamental to cell survival, cell development, tissue integration and coordination, and to the functional

viability of complex organisms. Within cutaneous tissue, keratinocytes interact with small populations

of other cell types including melanocytes, Langerhans cells, intra-epithelial lymphocytes and fibroblasts.

These interactions are critical to the multiple and diverse functions of the epidermis: a barrier to insult,

trauma and infection, environmental sensing and monitoring, moisture regulation, thermal regulation,

absorption, excretion, secretion, and immune defence [33-37]. In view of the evidence that EVs

contribute to the composition of extracellular matrix, pericellular and interstitial spaces, it is not

unreasonable to speculate that cutaneous activities are subject to modulation by interactions between

keratinocytes and EVs. The variety of potential signalling molecules; proteins, nucleic acids, glycans and

lipids; likely combine to modify the behaviour of recipient cells [38]. For example, melanocyte-

keratinocyte interactions are a well-studied system in which melanocyte activity such as pigment

production and pigment transfer, is facilitated through EVs (Fig 3) [3, 33]. In this context, keratinocyte-

derived EVs have been reported to regulate the expression and activity of tyrosinase, as well as

expression of the pigmentation-associated genes microphthalmia-associated transcription factor (MITF)

and Rab27a [3]. Importantly and to the points made above, the authors identified specific microRNAs

that were involved in this pathway including: hsa-miRNA-3196 and hsa-miR-203. It was found that hsa-

miRNA-3196 increased the expression of MITF-M and Rab27a, while hsa-miR-203 increased the

expression of tyrosine (TYR) and Rab27a increasing pigmentation [3]. Interestingly, these data

corroborate earlier work from Nogushi et al. who demonstrated tyrosinase expression and melanosome

transport were under the control of hsa-miR-203 [39]. Within the skin of mice, keratinocyte secreted EVs

are required to stimulate development of mature dendritic cells [40]. Keratinocyte-derived EVs also

increased the production of interleukin 6 (IL-6), IL-10 and IL-12 [40]. However, this observation does

not necessarily indicate specific immunity; when challenged with antigen (ovalbumin)-bearing EVs, T-

lymphocytes failed to induce antigen-specific T cell responses [40].

Cutaneous wound healing is a complex process of inter-dependent, overlapping events that combine to

repair skin tissue and restore skin function. With such complexity, effective and timely communication

between keratinocytes of the epidermis and fibroblasts in the dermis is critical to ensure that appropriate

temporal and spatial cellular events occur. Injury to the outermost epidermal layer stimulates fibroblasts

in the underlying layer to synthesize and secrete growth factors and signalling molecules, which in turn

feedback to stimulate keratinocyte proliferation, and differentiation [34]. It is well established that

keratinocytes and fibroblasts interact via integrins (ITG), heterodimeric cell-surface receptors

transducing outside-in and inside-out biochemical and mechanical cues between the extracellular matrix

(ECM) and the cytoplasm [41]. The ECM of the epidermis is enriched in EVs, released by primary human

epidermal keratinocytes, that include ITGα1, ITGα2, ITGα3, ITGα6, ITGβ1, ITGβ3, and integrin-

specific accessory molecules [24]. Interactions with integrins expressed by intact cells regulate multiple

cellular activities such as adhesion, migration, differentiation, apoptosis, and expression of specific genes

(Figure 2) [42, 43]. It is hypothesised that EVs secreted by keratinocytes and incorporated into ECM may

deliver their cargoes, including integrins, to dermal fibroblasts during healing processes [24]. Evidence

for the existence of “matrix-bound nanovesicles” (i.e. EVs) within natural ECM bioscaffolds was

recently reported [44]. Thus EVs embedded in ECM within the extracellular space may function as

anchors and facilitate cell-ECM adhesion. Keratinocyte-derived EVs are also known to transport

proteases, namely pro-MMP-1, MMP-3/TIMP-1 complexes, pro-MMP-8, and pro-MMP-9 [17, 24], and

thus have the potential to modify interstitial ECM ‘on demand’ in response to interactions with migrating

cells during injury and repair events. MMPs are involved in remodelling of extracellular matrix during

normal physiological processes, and are essential to wound reepithelialisation [45]. Notably, 14-3-3

protein σ and 14-3-3 protein β known to regulate MMP-1 activity, have been identified in cargoes from

keratinocyte-derived exosomes; thus matrix-bound EVs may be key enablers of cell migration during

tissue repair and regeneration [17, 46, 47]. This aspect of EV biology represents an opportunity for further

careful examination.

Proteases are just one population of EV cargo that may contribute to wound healing and tissue repair.

Mitogens and growth factors are also common components of the ECM, usually present as inactive pro-

forms or bound to ‘binding proteins’ that act as chaperones and prevent inappropriate activity [48, 49].

One mode of action is illustrated by the action of TGF-β1 on keratinocytes: keratinocytes interacting with

TGF-β1 are stimulated to secrete HSP90α-bearing EVs [1], and subsequently enhanced dermal fibroblast

and epidermal keratinocyte migration via LRP-1/CD91 cell surface receptors (Figure 2 and 3) [1]. TGF-

β1 is also a known suppressor of the phosphatase and tensin homologue (PTEN) [50-52], which, inhibits

the expression of type 1 collagen and connective tissue growth factor CCN2, thereby attenuating the

deposition of collagen into the pericellular space [53]. In association with EVs, EV- TGF-β1 is proposed

to indirectly affect collagen production through the regulation of microRNAs targeting PTEN [54]. These

data are interpreted to indicate that EV-TGF-β1 bound to ECM is presented to the integral transmembrane

receptor TGF-β1 receptor II (TGFβ1RII), prolonging the interaction and signalling cascade, resulting in

the expression of microRNAs that target PTEN thereby facilitating collagen expression [24, 54]. The

regulation of collagen expression by PTEN in wound sites is a potential mechanism to control the

sustained production of collagen and thereby attenuate the development of fibrosis and pathological

scars, hypertrophy and keloids. Therefore, EVs which can be engineered to carry miRNAs or molecules

that target PTEN may be effective for the treatment of skin pathologies related to collagen deposition.

Discussion

The active production and release of EVs by mammalian cells is recognised to be an important new mode

of cell-cell communication. Captured and stored in pericellular and interstitial spaces, EVs have a

demonstrated capacity to preserve bioactive cargoes packaged within lipid-enclosed vesicles,

transporting and delivering these cargoes to other cells. These include neighbouring cells, tissue resident

and transient immune cells, tissue stem cells and cells migrating from adjacent tissues in healing

responses to injury [5, 55]. While not intended to substitute for classical forms of intercellular

communication (e.g. hormones, chemokines, cytokines, mitogens), EVs provide local context,

potentially modifying the physiology and functional activities of cells in the immediate locale. While

there is a growing appreciation for the biogenesis and functional role of EVs, the mechanism(s) that

support the packaging of particular molecular information into EVs and the transmission of that

information to enable EV functionality remains uncertain. For instance, translocation and accumulation

of certain lipids to specific sites within the cellular plasma membrane seems to create membrane

asymmetry that induces the formation and release of EVs (biogenesis) [56-58]. This process has been

described for apoptotic bodies, microvesicles and exosomes [5, 59]. Similarly, the proposed mechanisms

through which EVs are thought to affect target cells include membrane fusion and internalization (direct

transfer of EV contents into target cells) and cell surface ligand/receptor interaction (no direct transfer of

EV content to target cells) [5]. However, an understanding of the molecular events and factors that trigger

the packaging of particular molecules that are found to be enriched in EVs is still limited [60, 61]. This

is despite there being evidence indicating that pathological conditions, stressors and trauma appear to

enhance the release of EVs that reflect specific cell physiology [13]. Thus the results from studies

designed to reveal more about the mechanism(s) that underlie the specific molecular packaging of various

EVs and the control of subsequent cell-to-cell communication through EVs will be of significant interest

to the field.

After release, EVs may randomly enter to blood vessels and freely circulate and systemically diffuse to

distant sites from the secreting cells, such as intestine and brain [62-64]. Of note, EVs seem to

preferentially target cells depending on antigen-presentation that match EVs and target cells [65]. For

example, exosomes bearing TCR/CD3 on their surface appear to target cells bearing specific peptide-

MHC combinations [66]. On the other hand, oligodendrocyte-derived EVs are preferentially internalised

in microglia that do not seem to have antigen-presenting capacity [67]. In addition, to natural specific

cell targeting by EVs, recently, EVs bearing engineered peptides on the surface have been generated in

order to specifically target cells or tissues [68, 69]. However, up to date there are no reports of any

preferential cell targets of keratinocyte-derived EV’s. If the understanding of keratinocyte-derived EV

targets increases in the future, then such EVs might be able to be targeted to wound healing or other skin

diseases.

Conclusion

The capacity of EVs to deliver bioactive cargoes also suggests that EVs could act as potential vehicles

in which to deliver therapeutic reagents. The demonstrated function of keratinocyte-derived EVs in vitro

includes stimulation of skin cell mediated ECM remodelling, cell migration and differentiation, pigment

production, and facilitating wound healing. Studies have reported the physiological conditions under

which EVs can be produced, and enriched with specific molecules thus potentially reflecting the

pathological state of the parental cells [5]. Evidence for the transfer of keratinocyte-derived EVs

containing multiple bioactive species between skin cells by active or passive mechanisms, are strong

indicators for potential similar functions in vivo. As a consequence, there is growing interest in the

therapeutic application of EVs. The potential to manufacture clinical grade EVs containing therapeutic

cargoes remains to be demonstrated, although current technologies on engineering and collecting EVs

suggest this possibility is achievable [69, 70]. Clearly further laboratory studies defining the clinical

potential that EVs hold will be followed by the preclinical and clinical validations that are required to

translate EV technologies into applications in the real world.

Figure 1: Summary schematic of keratinocyte-derived EVs modulation of pigmentation by melanocytes

[3]. Keratinocyte-derived EVs carrying miRNA-203 and miRNA-3196 increase pigmentation by two

different mechanisms. EV-miRNA-203 increases the production of tyrosine (TYR) and Rab27a proteins

which then induces TYR activity and therefore up-regulation of pigmentation. On the other hand, EV-

miRNA-3196 increases the production of microphthalmia-associated transcription factor (MITF)

melanocyte isoform (MITF-M) and Rab27a which then activates the MITF pathway to up-regulate

melanin production.

miR-203 miR-3196

Keratinocyte-derived EV

EV-miR-203

EV-miR-3196

!MITF-M !Rab27a

MITF pathway

Melanin production

!TYR !Rab27a

TYR signaling

Nucleus

Figure 2: Summary schematic of mechanisms through which keratinocyte-derived EVs modulate target

fibroblasts [2, 14, 17]. (1) EVs in conditioned medium carry HSP90� that interacts with LPR1/CD9

receptor on the surface of fibroblasts and results in increased fibroblast migration. (2) EVs fuse directly

with the fibroblast membrane, are internalised and activate ERK1/2, JNK, p38, Smad3 and TGF- β1

which further influences gene expression and protein production. These are thought to increase fibroblast

migration, differentiation and formation of basement membrane and thereby wound healing. (3)

Keratinocyte-derived EVs not internalised by fibroblasts ‘decorate’ the extracellular matrix (ECM)

which enables subsequent cell migration and wound healing.

Figure 3: Summary schematic of keratinocyte-derived EV autocrine activity [1, 12, 18, 19]. EVs released

by keratinocytes following specific stimuli carry various molecules that influence several biological

processes. (A) Gama irradiation of keratinocytes stimulates the secretion of EVs that signal in an

autocrine manner which increases bystander signalling, reactive oxygen species (ROS) and calcium;

consequently, leading to an increase in cell death. (B) Keratinocytes subjected to serum starvation,

secrete EVs that present Cathepsin B on their membrane surface which binds with cathepsin B-specific

plasma membrane receptors on target keratinocytes leading to extracellular matrix (ECM) remodelling

and cell migration. (C) Keratinocytes subjected to hypoxia secrete EVs carrying HSP90� which can

interact with LPR1/CD9 receptor on the keratinocyte surface and increase keratinocyte migration.

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Table 1: Summary of keratinocyte-derived EVs, their target cells and bioactivity

EV type Parental cell Stimuli

Functional molecules

presented in EVs

Target molecule Target cell Bioactivity References

CM Migrating HaCaT - Cathepsin B - HaCaT

Stimulating wound healing in HaCaT monolayer

Buth et al., 2004[12]

Exosome, CM Human foreskin keratinocyte Serum free Stratifin MMP-1 Fibroblast

Stimulating MMP-1 expression in fibroblasts

Chavez-Munoz et al., 2008[2]

Exosome, CM

Human differentiated keratinocyte, undifferentiated foreskin keratinocyte Serum free

50 cytoplasmic proteins, 14-3-3 family ECM

Dermal fibroblast

Working as ECM modulating factors for dermal fibroblasts, stimulating MMP-1 expression in fibroblasts

Chavez-Munoz et al., 2009[17]

Exosome, microvesicle HaCaT Irradiation -

Bystander Signalling

Human keratinocytes, HaCaT

Decreasing cell viability, increasing ROS production, involving in the calcium signalling

Jella et al., 2014[18]

Microvesicle

Normal epidermal keratinocyte, HaCaT Serum free - -

Dermal fibroblast, endothelial cells

Regulating the gene expression and protein production, activating ERK1/2, JNK, Smad, and p38 signalling pathways in fibroblasts, stimulating

Huang et al., 2015[14]

fibroblast migration and inducing tube formation

Exosome Human foreskin keratinocytes Irradiation

hsa-miR-3196, has- miR-203 -

Human foreskin neonatal melanocytes

Modulating the activity of tyrosinase (TYR) leading to stimulate pigmentation

Lo Ciceroet al., 2015[3]

Exosome Human foreskin keratinocytes

Serum depleted EV medium, poly(I:C) stimulation IL-36γ - -

Modulating both local and systemic immune responses to viruses and other pathogens

Rana et al., 2015[15]

CM Human neonatal keratinocytes TGFα HSP90α

LRP-1/CD91 receptor

Human keratinocytes, dermal fibroblasts

Promoting human keratinocyte and dermal fibroblast migration

Cheng et al., 2008[1]

Apoptotic body, microvesicle, exosome

Human dermal keratinocyte, HaCaT Serum free

581 miRNAsfrom HaCaT, 508 miRNAsfrom dermal keratinocyte - -

Associated with different biochemical pathways

Than et al., 2018[16]

CM

Primary human neonatal keratinocyte Hypoxia HSP90α -

Human keratinocyte

Increasing human keratinocyte migration

Woodley et al., 2009[19]

CM: Conditioned media=

Table 2: Expression of 14-3-3 protein in exosomes released by differentiated and undifferentiated keratinocytes. 14-3-3 isoform

Undifferentiated keratinocyte

-derived exosomes

Differentiated keratinocyte

-derived exosomes

σ - +

β + +

η ++ +

ε - +

ζ + +

γ + +

τ - +

(-) no expression; (+): expression level of protein; (++): higher expression level of protein !