Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 2, pp. 289-298 © Pleiades Publishing, Ltd., 2025.

Published in Russian in Biokhimiya, 2025, Vol. 90, No. 2, pp. 321-331.

289

Induction of Fibroblast-to-Myofibroblast

Differentiation by Changing Cytoplasmic Actin Ratio

Yulia G. Levuschkina

1,2#

, Vera B. Dugina

1,2#

, Galina S. Shagieva

Sergey V. Boichuk

3,4

, Ilya I. Eremin

, Natalia V. Khromova

, and Pavel B. Kopnin

6,a

Belozersky Research Institute of Physico-Chemical Biology,

Lomonosov Moscow State University, 119992 Moscow, Russia

Faculty of Biology, Lomonosov Moscow State University, 119991 Moscow, Russia

Department of Pathology, Kazan State Medical University, 420012 Kazan, Russia

Department of Radiotherapy and Radiology,

Russian Medical Academy of Continuous Professional Education, 119454 Moscow, Russia

Petrovsky National Research Center of Surgery, 119991 Moscow, Russia

Scientific Research Institute of Carcinogenesis,

N.N. Blokhin National Medical Research Center of Oncology, 115478 Moscow, Russia

e-mail: pbkopnin@mail.ru

Received November 19, 2024

Revised January 9, 2025

Accepted January 14, 2025

Abstract— Myofibroblasts, which play a crucial role in the tumour microenvironment, represent a promising

avenue for research in the field of oncotherapy. This study investigates the potential for the induced differ-

entiation of human fibroblasts into myofibroblasts through downregulation of the γ-cytoplasmic actin (γ-CYA)

achieved by RNA interference. A decrease in the γ-CYA expression in human subcutaneous fibroblasts resulted

in upregulation of myofibroblast markers, including α-smooth muscle actin (α-SMA), ED-A FN, and type  III

collagen. These changes were accompanied by notable alterations in cellular morphology, characterized by

a significant increase in cell area and the formation of pronounced supermature focal adhesions. Downreg-

ulation of γ-CYA resulted in the compensatory increase in expression of the β-cytoplasmic actin and α-SMA,

and formation of the characteristic α-SMA-positive stress fibers. In conclusion, our results demonstrate that

a decrease in the γ-CYA expression leads to myofibroblastic trans-differentiation of human subcutaneous

fibroblasts.

DOI: 10.1134/S000629792460412X

Keywords: actin isoforms, fibroblasts, myofibroblasts, stromal microenvironment, differentiation

Abbreviations: α-SMA, α-smooth muscle actin; β-CYA, β-cytoplasmic actin; γ-CYA, γ-cytoplasmic actin; CAMs, cancer-

associated myofibroblasts; ECM, extracellular matrix; ED-A FN, fibronectin extra domain A; FAs, focal adhesions; IF, im-

munofluorescence; shRNA, short hairpin RNA; SMM, smooth muscle myosin.

* To whom correspondence should be addressed.

These authors contributed equally to this study.

INTRODUCTION

Actin is represented by six different isoforms in

higher vertebrates, two of which, β- and γ-cytoplas-

mic actins (β-CYA and γ-CYA), are ubiquitously ex-

pressed in all cell types. The ratio of these two iso-

forms has been demonstrated to influence phenotypic

properties and functional activities of the cells [1].

The study of intracellular localization and functional

roles of β-CYA and γ-CYA became feasible following

development of monoclonal antibodies highly specific

to γ-CYA. The use of polyclonal antibodies is limited

due to the potential for cross-reactivity between γ-CYA

and α-smooth muscle actin (α-SMA) [2]. Researchers

LEVUSCHKINA et al.290

BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025

have attempted to determine the role of cytoplasmic

actin isoforms by selectively inhibiting expression of

each using short hairpin RNA (shRNA). Decrease of the

β-CYA expression in fibroblasts led to an increase in

cell surface area, formation of multiple protrusions,

and disassembly of stress fibers, while the downregu-

lation of γ-CYA resulted in the acquisition of a “contrac-

tile phenotype” with well-defined actin bundles [2, 3].

Furthermore, fibroblasts with reduced expression of

β-CYA or γ-CYA exhibited distinctive patterns of cell

motility in comparison with the control cell culture,

indicating that both cytoplasmic actin isoforms could

have a specific role in regulating cell motility [2, 4].

Since their initial identification, myofibroblasts

attracted significant attention within the scientific

community, having been implicated in a multitude

of pathological processes, including those associated

with tumour progression. The designation “cancer-

associated myofibroblasts” (CAMs) is frequently used

to describe this specific type of myofibroblasts. The

role of CAMs in tumour progression is complex and

multifaceted. In particular, myofibroblasts initially ex-

ert an anti-tumour effect, but subsequently become

activated by factors secreted by the tumour and con-

tribute to its growth and progression [5]. CAMs are

the primary source of extracellular matrix (ECM) com-

ponents that facilitate tumour cell growth and pro-

gression. These include matricellular proteins such

as connective tissue growth factor (CTGF), tenascin  C,

fibronectin, collagens, and elastin [6].

Myofibroblasts associated with tumour stroma

can be activated from different progenitor cells. Un-

der normal conditions, progenitor cells in the intact

tissue are protected by an extracellular matrix of a

specific composition, which prevents development

of enhanced contractile properties. In the event of

a disruption of tissue homeostasis, the release of in-

flammatory signals activates stromal cells to remodel

ECM. This results in a gradual increase of stiffness,

thereby facilitating formation of contractile bundles

of microfilaments, which are known as stress fibers.

This phase is characterized by generation of protomy-

ofibroblasts. Subsequently, transforming growth fac-

tor (most often TGFβ1), which is a chemical factor, in

combination with a rigid ECM, which is a mechanical

factor, stimulates expression of α-SMA by protomy-

ofibroblasts and incorporation of this actin isoform

into stress fibers. This results in further remodelling

of ECM. The combination of physicochemical factors

originating from protomyofibroblasts and differenti-

ated myofibroblasts facilitates epithelial cell transfor-

mation and tumour progression [6, 7].

The primary marker of myofibroblasts at the

present time is α-smooth muscle actin (α-SMA) iso-

form, which has been identified in stress fibers with

distinctive contractile properties  [5]. Protomyofibro-

blasts are distinguished by the formation of stress

fibers composed of β-CYA [8].

Clarification of myofibroblast cytogenesis is inex-

tricably linked to the challenge of their identification.

These cells could be distinguished from fibroblasts by

their capacity for contraction, presence of organized

bundles of microfilaments, and their interaction with

matrix and other cells. However, similar cell types,

such as smooth muscle cells (SMC), could also exhibit

similar functional properties and, therefore, be con-

sidered to align with this description [9].

Hence, the necessity for identification of partic-

ular molecular markers that can be used to distin-

guish between the closely related cell types is evident.

The initial assumption that α-smooth muscle actin is

an optimal molecular marker for this purpose, which

is based on the fibroblast-myofibroblast differenti-

ation pathway, is ambivalent in the case when SMC

or myoepithelial cells are present in close proximity

to putative myofibroblasts  [10]. Furthermore, studies

have indicated the potential for interchangeability be-

tween the different actin isoforms. Nevertheless, this

does not imply that α-smooth muscle actin should be

discarded as a marker for identification purposes [11].

It is sufficient to introduce additional parameters to

enhance accuracy of identification.

In the present study, we investigated the potential

of fibroblast differentiation into myofibroblasts, with

the objective of elucidating the role of the cytoplasmic

actin ratio in this process. Using the previously cre-

ated lentiviral genetically engineered constructs with

proven efficacy [12,  13], we partially or completely

downregulated γ-CYA and performed comparative

morphometric analyses with the control at different

time points. Efficiency of the γ-CYA downregulation

was evaluated through the determination of the quan-

tity of mRNA using classical PCR analysis, measure-

ment of the amount of protein (immunoblotting), as

well as observation of morphological alterations and

calculation of the percentage of cells exhibiting down-

regulation of γ-CYA (phase-contrast and immunofluo-

rescence microscopy).

MATERIALS AND METHODS

Cell culture. Fibroblast cell cultures were ac-

quired from the culture collection of the cytogenet-

ics laboratory of the Scientific Research Institute of

Carcinogenesis, N.  N.  Blokhin National Medical Re-

search Center of Oncology. The cells were cultivated

in a DMEM-HiGluc medium with Glutamax (glutamine

was present in the medium) and NEAA (a mixture of

essential amino acids).

RNA interference. The most efficient 21-bp

sequence was selected for shRNA expression and

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BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025

inhibition of γ-CYA mRNA at position 1790-1811:

5′-CAGCAACACACACGTCATTGTGTGTAA-3′. The corre-

sponding constructs were synthesized and cloned

into the lentiviral vector pLKO.1-puro (Addgene, USA;

Plasmid #10878). A control was also included in the

form of the pLKO.1-shGFP-puro construct containing

a shRNA sequence targeting eGFP (GenBank pEGFP

accession number U55761) was used as a control.

Oligonucleotide synthesis and DNA sequencing were

conducted by Evrogen.

The lentiviral DNA constructs pLKO.1, along with

the packaging plasmids pΔR8.2 (#12263, Addgene) and

pVSV-G (#8454, Addgene), were transfected into 293FT

packaging cells (R70007, Thermo Fisher Scientific,

USA) using TurboFect transfection reagent (R0531,

Thermo Fisher Scientific).

Supernatants containing the virus were collected

after 1-2 days of incubation and used to infect fibro-

blasts in the presence of polybrene (8  μg/ml, Sigma-

Aldrich, USA). The infected cell cultures were main-

tained for 4-5 days in a medium containing 1  μg/ml

puromycin (Sigma-Aldrich). The cells were used in

experiments 5-9 days after the onset of infection.

Antibodies. The following primary antibodies

were used: mouse monoclonal antibodies – anti-α-SM1

(1A4, IgG2a, AbD Serotec, UK), anti-γ-CYA (2A3, IgG2b,

AbD Serotec), anti-β-CYA (4C2, IgG1, AbD Serotec),

anti-pan-actin (clone C4, Cell Signaling Technology,

USA), anti-paxillin (IgG1, BD Transduction Laborato-

ries, USA), anti-desmin (BD Transduction Laborato-

ries); rabbit monoclonal antibodies – anti-collagen  I,

anti-elastin and anti-collagen III (AbD Serotec); rabbit

polyclonal antibodies– anti-SMM (smooth muscle my-

osin) antibody (Biorad). Fibronectin extra domain  A

(ED-A FN) was stained with the IST-9, mouse mono-

clonal antibody IgG1 specific to the type  III domain

(kindly provided by Dr.  L.  Zardi, National Institute for

Cancer Research, Laboratory of Cell Biology, Genoa,

Italy).

The following secondary antibodies were em-

ployed: goat anti-mouse IgG H&L (Alexa Fluor® 488,

Subclass1/Subclass2b), goat anti-mouse IgG H&L (Alexa

Fluor® 594, Subclass 2b), donkey anti-rabbit IgG H&L

(Alexa Fluor® 488), Rhodamine RedTM-X-conjugated

donkey anti-mouse IgG H&L (Jackson ImmunoResearch

Laboratories, UK). DAPI (D9542, Sigma-Aldrich) was

used for nuclear staining during incubation with the

secondary antibodies. For the purpose of Western blot

analysis, HRP-conjugated secondary antibodies were

used, namely anti-mouse IgG and anti-rabbit IgG

(Santa Cruz Biotechnology, USA).

Western blot analysis. Cell cultures were lysed

on plastic dishes in a RIPA buffer (50  mM Tris-HCl

pH  7.4, 150  mM  NaCl, 1%  Na  deoxycholate, 1%  NP-40,

0.1%  SDS, 100  mM  PMSF, 1  mM  pepstatin  A and

1  mM  E64) at 4°C to obtain extracts. Protein concen-

tration in cell lysates was measured using the Brad-

ford assay. Protein samples containing from 5 to 20  μg

were separated in an 8-12% polyacrylamide gel with

SDS and transferred to a PVDF membrane (IPFL00010,

Millipore).

Membranes were blocked with a SuperBlock Block-

ing Buffer (Thermo Fisher Scientific) for 15-20  min at

room temperature and next stained with specific an-

tibodies for 1  h at room temperature. The membranes

were then washed three times for 5  min each with

ice-cold PBS with Tween20 (PBS-T) on a shaker. Second-

ary antibodies were next added and incubated with

the membranes followed by another PBS-T wash three

times for 5min each with ice-cold PBS-T. Finally, after

pre-drying, the bands were visualized using enhanced

chemiluminescence WesternBright Quantum detection

kit (Advanstra, USA). Densitometric analysis of West-

ern blotting images was performed using TotalLab

software (version 1.11) and normalized to total actin

levels. Protein levels were estimated by analyzing the

results of at least three independent experiments.

Fluorescent immunohistochemistry and immu-

nofluorescent microscopy. For cell fixation, 2%para-

formaldehyde was used in a serum-free culture me-

dium with addition of HEPES (1  ml of 1  M  HEPES

was added per 50  ml of serum-free medium). The

fixation period was between eight and ten minutes.

Subsequently, the cells on coverslips were subjected

to an extraction-fixation process with cold methanol

(1  ml per well of a six-well plate) at −20°C for a peri-

od of five minutes. Following washing, the cells were

stained with antibodies in order to detect cytoskeletal

structures in accordance with the established proto-

cols. Immunofluorescence examinations were con-

ducted using a Zeiss Axioplan microscope, equipped

with an Olympus DP70 video camera. Two objectives

were employed: a 40×/0.75 PlanNeofluar and a Plan-

Neofluar 100×/1.3.

Morphometrical and statistical analyses. ImageJ

Fiji v1.53u software was used for tracing outlines of

the cell, measurement of the cell area, and mean of

IF intensity of cytoskeletal proteins per cell. The data

were then subjected to further processing with Adobe

Photoshop Version: 22.4.2 (2021). The results are pre-

sented as a mean ± standard error of the mean ob-

tained from at least three independent experiments.

The results were subjected to statistical evaluation us-

ing the Mann–Whitney U-test. Values of p  <  0.001  (***),

p  <  0.01  (**) and p  <  0.05  (*) were considered to be sta-

tistically significant.

Three square areas with dimensions of 10×10  μm

(100  μm

) in the lamellar zone of each cell edge, cor-

responding to locations of the highest total fluores-

cence of focal adhesions (FAs), were selected for mea-

surement of intensity values. As our objective was to

examine changes in the morphometric parameters of

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BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025

Fig.  1. Cultures of subcutaneous fibroblasts (a) and dental pulp fibroblasts (b) 5 days and 9 days after infection with

lentiviral constructs, IF staining for α-SMA (green) and γ-CYA (red). Scale bar is 50 μm. c) IF intensity of α-SMA in the

α-SMA-positive cells in subcutaneous (left) and dental pulp (right) fibroblasts. Graphs show mean ± SEM. d) Amount of

α-SMA-positive cells in the subcutaneous fibroblast cultures in the control and in the cells with downregulated γ-CYA, 5days

and 9 days after infection. Graphs show mean ± SEM, the mean was calculated based on a minimum of 3 independent

experiments, with at least 100 cells analyzed in each experiment. Mann–Whitney U test was used for statistical analysis

for all comparisons. ** p < 0.01 for all panels.

thecontacts themselves, rather than distribution with-

in the cell, a limited area was selected in high-magni-

fication images. This approach enabled us to extract

three independent areas for measurements and to

document more detailed changes in these parame-

ters. Subsequent measurements, based on manual

threshold adjustment in the ‘Analyze Particles’ tool,

were conducted to determine number and area of FAs.

At least 30 cells from three experiments were exam-

ined in each group. No significant differences were

observed between the areas of the identical groups

selected in the different experiments. The obtained

values were summarized separately for each control

and experimental group. Subsequently, the corre-

sponding groups of cell contacts were distinguished by

the size of the area occupied by a single focal contact.

On the basis of the morphometric measurements for

each type of the cell culture, the FAs were classified

into three categories: immature (area ≤2  μm

), ma-

ture (area~2-6  μm

), and supermature (area ≥6  μm

Finally, the average percentage of FAs for three ex-

periments was presented as the distribution of the

contacts with different areas.

RESULTS

Human subcutaneous fibroblasts were chosen

as an optimal cell culture. The objective of this study

was to examine the mechanisms of induced differ-

entiation. For this purpose, subcutaneous fibroblasts

and dental pulp fibroblasts from healthy donors were

used. Expression of the specific markers was subse-

quently analyzed in both samples after culturing of

the treated cells for varying periods.

In the course of experiments conducted with

different fibroblast cultures, identification of α-SMA

(primary marker of myofibroblasts) revealed that

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BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025

Fig.  2. a)  IF staining for γ-CYA, β-CYA, α-SMA in human subcutaneous fibroblasts 9 days after shRNA treatment, scale

bar – 25 μm. b)  γ-CYA, β-CYA, and α-SMA fluorescence intensity in subcutaneous fibroblasts 9 days after infection. Graphs

show mean ± SEM. c)RNA profiling data, given in relative units for human subcutaneous fibroblasts 9 days after RNA inter-

ference. ACTA2 – gene of α-smooth muscle actin, ACTB – gene of β-cytoplasmic actin, ACTG1 – gene of γ-cytoplasmic actin.

d, e) WB analysis of actin isoforms in human subcutaneous fibroblasts with downregulated γ-CYA. Graphs show relative

levels of actin isoforms (mean ± SEM), the mean was calculated based on a minimum of 3 independent experiments, with

at least 100 cells analyzed in each experiment. Mann–Whitney U-test was used for statistical analysis for all comparisons.

**  p  <  0.01, ***  p  <  0.001 for all panels.

themore efficient differentiation was observed in the

culture of subcutaneous fibroblasts (Fig.  1a). On the

contrary, the same processes occurred at a significant-

ly slower rate in the dental pulp fibroblasts (Fig.

  1b).

Downregulation of γ-CYA was more effective in the

case of subcutaneous fibroblasts, resulting in a higher

percentage of the cells with activated myofibroblast

phenotype (Fig. 1c).

Furthermore, it was determined that the optimal

period for observation of induced differentiation is

9 days, as structural changes in the cytoskeleton and

changes in expression of the corresponding actin iso-

forms were significantly more pronounced than those

observed at the day 5 post-infection (Fig. 1d).

However, the use of dental pulp fibroblasts with

reduced proliferative activity offers a significant

advantage for the detection of specific markers. In

particular, organization of some proteins of focal ad-

hesions, such as paxillin, requires long-term contin-

uous cultivation of the cells on the same substrate.

Consequently, their highest expression would be ob-

served in the dental pulp fibroblasts at a later point

in time.

A decrease of γ-CYA expression was accompa-

nied by an increase of the β-CYA and α-SMA expres-

sion. In the course of investigation of fibroblasts with

downregulated γ-CYA, it was observed that a change

in the amount of one actin isoform was compensated

by a reciprocal change in the other. Specifically, a de-

crease in the level of γ-CYA was accompanied by an

increase in the expression of α-SMA and β-CYA in all

infected cells (Fig. 2a). Therefore, the overall quanti-

ty of actin (pan-actin) remained unaltered. Such com-

pensation was observed at both mRNA and protein

levels. However, while γ-CYA was almost completely

downregulated at the mRNA level (Fig.  2c), less signif-

icant downregulation was observed in the WB analy-

sis (Fig.  2, d, e) and IF staining (Fig. 2b). This suggests

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BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025

Fig.  3. Upregulation of myofibroblast markers in human subcutaneous fibroblasts after γ-CYA downregulation. a)  IF staining

for ED-A FN (red, scale bar – 50  μm) and collagen  III (green, scale bar – 25  μm), control in comparison to shRNA against

γ-CYA (9  days after infection). b)  ED-A FN and collagen  III fluorescence intensity (9  days after infection), presented in arbi-

trary units. Graphs show mean ± SEM. c)  Changes in the cell area (μm

) of the fibroblasts in control and in the cells with

downregulated γ-CYA (5 days after infection). d)  Formation of stress fibers 5 days after γ-CYA downregulation. IF stain-

ing for α-SMA (green), DAPI (blue) was used for DNA (nuclear) staining. Scale bar – 25 μm. e)  Formation of a nuclear-

associated actin network in the fibroblasts with downregulated γ-CYA (5 days after infection). IF staining for γ-CYA (red)

and α-SMA (green), DAPI (blue) was used for DNA (nuclear) staining. Scale bar – 25  μm. Mann–Whitney U-test was used

for statistical analysis for all comparisons. **  p  <  0.01 for all panels. The mean was calculated based on a minimum of

3 independent experiments, with at least 100 cells analyzed in each experiment.

the existence of a complex post-transcriptional rear-

rangement in the case of different actin isoforms.

Evidence for effectiveness of the induced differ-

entiation of fibroblasts into myofibroblasts follow-

ing γ-CYA downregulation. To confirm the presence

of induced differentiation, the most commonly used

cytoskeletal marker proteins were selected as positive

controls, specifically α-SMA and ECM proteins such

as ED-A FN, elastin, and collagens of various types.

The measured IF intensity of ED-A FN and type

III collagen in the control group and in the fibroblasts

with downregulated γ-CYA allowed us to reveal a sub-

stantial increase in the expression of these markers in

the activated cells (Fig. 3 a and b).

Changes in the fluorescence intensity of elastin

in the control and in the culture with downregulated

γ-CYA were not statistically significant. The observed

decrease in typeI collagen in the γ-CYA-depleted cells

was contrary to the theoretical expectations, and

could probably be associated with the processes re-

lated to the timing of cell differentiation in culture

(data not shown).

The results of the IF staining assay for paxillin

will be discussed in detail in the following section,

which is related to maturation of the focal adhesions.

The success of differentiation of fibroblasts into

myofibroblasts was further confirmed by the negative

control markers – SMM and desmin. These markers

are characteristic of smooth muscle cells and absent

in myofibroblasts. In the presence of α-SMA, a com-

mon marker for both cell types, there was no staining

of SMM and desmin in the fibroblasts with downreg-

ulated γ-CYA (data not shown). Morphometric analy-

sis of positive and negative markers of myofibroblasts

enabled us to determine the precise direction of the

induced differentiation.

Downregulation of γ-CYA expression in human

subcutaneous fibroblasts resulted in an increase in

the cell area. Differences in the morphology of control

fibroblasts and fibroblasts with downregulated γ-CYA

were already evident when observed by phase-con-

trast microscopy (data not shown). Control fibroblasts,

particularly those lacking α-SMA, had an elongated

shape with long tails (sometimes spindle-shaped) and

did not have prominent stress fibers. The fibroblasts

with downregulated γ-CYA had a polygonal shape,

a significantly larger cell area, and more flattened

edges on the surface.

Areas of the cells with downregulated γ-CYA ex-

pression were significantly larger than those in the

control group (Fig.3c). This was observed even in the

cells that did not have time to develop a prominent

actin network after infection with lentiviral constructs

due to insufficient differentiation time (Fig. 3c, left).

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BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025

Fig.  4. a)  IF staining for paxillin in human subcutaneous fibroblasts 9 days after infection investigated in the control and

in the cells with downregulated γ-CYA. Scale bar –10  μm. b)  Dynamics of FAs maturation 5 and 9 days after infection with

lentiviral constructs. c)  Histograms of distribution of FAs with different areas, 5 and 9 days after infection, control com-

pared to the cells with downregulated γ-CYA. FAs of at least 30 cells were calculated per experiment, based on the data

from at least three independent experiments.

It is noteworthy that a certain percentage of

α-SMA-positive cells was observed in the control cul-

ture, although this was significantly lower than in the

γ-CYA-depleted cells (Fig.  3c, right). In order to clas-

sify the cells as α-SMA-positive or α-SMA-negative, it

was necessary to consider not only the intensity of

the corresponding IF staining, but also the structur-

al organization of the actin network. In particular, in

the cells with downregulated γ-CYA, formation of the

characteristic actin bundles or stress fibers (Fig.  3d)

and specific actin network associated with the nucleus

or near-nuclear (Fig. 3e) were observed.

Myofibroblastic transition was accompanied by

maturation of the focal adhesions. Focal adhesions

(FAs), specialized contact sites between the cells and

ECM, play an important role in mechanotransduction,

a process that stimulates myofibroblast differentiation,

which, as we can see from the previous section, occurs

better in the fibroblasts with downregulated γ-CYA.

To evaluate the dynamics of focal adhesion matura-

tion, IF staining for paxillin was used (Fig. 4a). Esti-

mates of the number of FAs per 100  µm

area and their

area measurements in the control and γ-CYA down-

regulated cells are presented in the Tables (Fig. 4b).

To evaluate the degree of maturation at different stag-

es, cell cultures were observed following infection with

lentiviral constructs for 5 and 9 days (Fig. 4b).

The majority of the FAs observed in the cells from

the control cultures were immature, although a sig-

nificant number of such FAs were also present in the

cells with downregulated γ-CYA. The number of ma-

ture focal contacts was higher in the cells with down-

regulated γ-CYA, although they were also present in

relatively small numbers in the control cells. Forma-

tion of the supermature contacts was observed exclu-

sively in the cells with downregulated γ-CYA (Fig.4c).

In conclusion, the results of morphometric analy-

sis demonstrate that differentiation of fibroblasts into

myofibroblasts was accompanied by the process of

maturation or fusion of FAs. This is evidenced by a

decrease in the number of individual contacts, accom-

panied by an increase in their area.

DISCUSSION

Actin is a highly conserved protein and the great-

est divergences are observed when the muscle and

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BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025

cytoplasmic isoforms are compared. Amino acid res-

idues important for formation of the fibrillar actin

are mostly identical, and the major differences in the

amino acid composition are concentrated in the N-ter-

minal region, outside of the subdomain

  1. In particu-

lar, β-CYA and γ-CYA differ by only four amino acid

residues in this sub-region [14, 15]. Despite the fact

that actin isoforms exhibit a high degree of similarity

in terms of their amino acid sequences, there are nev-

ertheless notable differences in their localization and

functional roles within the cell. β-CYA is predominant-

ly present in stress fibers, whereas γ-CYA is detected

mainly as an apical dense network [2]. Some studies

have indicated that minor variations in the N-terminal

sequence of β-CYA and γ-CYA may affect their affinity

for actin-binding proteins (ABPs) [16].

While the degree of difference between β-CYA

and γ-CYA at the amino acid level is only 1%, it ex-

ceeds 11% for the corresponding mRNAs. In addition,

the β-CYA mRNA is initially synthesized at a rate six

times higher than that of γ-CYA. However, no such

significant difference is observed at the protein level,

apparently indicating that the majority of β-CYA tran-

scripts are repressed and stored for potential rapid

translation of the corresponding protein and dynamic

rearrangement of the cytoskeleton [1]. The observed

discrepancies in the mRNA and protein levels could be

explained by the fact that β-CYA is a more dynamic

isoform that is critically required by the cell for cyto-

skeletal rearrangement. Taking into account the fact

that we used adult donor fibroblasts, degradation of

the already translated γ-CYA protein was slower and

the effect of silencing was significantly less at the pro-

tein level, despite the expected drastic decrease in the

mRNA level [1, 17].

Myofibroblasts are of heterogeneous origin, which

means that they are derived from the different types

of precursors via appropriate processes, with fibro-

blast differentiation representing only one potential

pathway [18]. However, properties of the fibroblasts

themselves could vary depending on their localization.

The more efficient differentiation of the subcutaneous

fibroblasts has been observed for a reason: this cell

type is inherently richer in the relevant markers, as

their localization implies greater dynamics and suscep-

tibility to mechanotransduction – a phenomenon that

determines the dependence of myofibroblast differen-

tiation on the changes in mechanical stress caused by

both exogenous and endogenous factors. This applies

not only to the organization of the fibroblast cytoskel-

eton, but also to the synthesis of ECM proteins [19].

In particular, these features are directly reflected in

the organization of the cell–cell contacts, which in-

clude some of the markers we have investigated.

Despite the established role of ED-A FN in myofibro-

blast differentiation, the mechanism underlying this

process remains unclear. However, it has been suggest-

ed that ED-A FN could interact with the fibroblast cell

surface receptors and participate in the TGFβ1-mediat-

ed signaling pathway. Indeed, the data obtained from

RNA profiling (data not shown) indicate an increase

in the TGFB2 and corresponding TGFBR2 (TGFβ re-

ceptor

  2) transcripts. At the same time, the level of

TGFBR3 (TGFβ receptor  3) transcript is reduced by ap-

proximately 20%, suggesting that co-reception may be

redundant under the new differentiation conditions.

Accumulation of ED-A FN is frequently observed in the

cases of fibrosis. Unique expression of ED-A FN and

its key role in the development of the myofibroblast

phenotype makes this protein an attractive target for

anti-fibrotic therapy [20,  21]. Morphometric changes

observed in this study, including changes in the cell

area and shape, are consistent with the previously

published data [22,  23]. We suggest that downregula-

tion of γ-CYA induces alterations in the expression of

ECM proteins, increases microenvironment stiffness,

and thus stimulates the formation of supermature

FAs, which are associated with mechanotransduction.

Asaresult, we observe transition of fibroblasts to the

protomyofibroblast state [24, 25].

The observed involvement of γ-CYA in the phe-

notypic switching could be attributed to its normal

physiological functions in the cytoskeletal rearrange-

ments of the developing cells. Previously, it has been

demonstrated that suppression of the Rho-kinase-

dependent cell migration is associated with a decrease

in γ-CYA expression during neuroblastoma differenti-

ation [26]. In addition, γ-CYA inhibits myofibroblastic

cell trans-differentiation through two complementary

mechanisms: by regulating the G-/F-actin ratio and by

binding to the myocardin-related transcription/serum

response factor (MRTF/SRF) complex. Importantly, it

is γ-CYA that preferentially binds to MRTF-A and SRF,

resulting in the appropriate transcription program be-

ing initiated upon depletion of this isoform [27].

CONCLUSION

In the course of our investigation, we established

experimental conditions for the induced differentia-

tion of fibroblasts into myofibroblasts through the

downregulation of γ-CYA expression (Fig. 5). On the

basis of the morphometric data and of the marker

expression profile, human subcutaneous fibroblasts

were identified as an optimal cell culture for our re-

search. A decrease in the γ-CYA expression was accom-

panied by a compensatory increase in the expression

of other actin isoforms. Changes were revealed at the

mRNA level, at the protein level, and at the level of

immunofluorescence signal. The fibroblasts with ful-

ly or partially downregulated γ-CYA had an increased

RATIO OF CYTOPLASMIC ACTINS INFLUENCES DIFFERENTIATION 297

BIOCHEMISTRY (Moscow) Vol. 90 No. 2 2025

Fig. 5. Scheme illustrating the features of the fibroblast-to-my-

ofibroblast differentiation induced by γ-CYA downregulation.

area, distinct stress fibers, mature and supermature

FAs, which are typical of the myofibroblast phenotype.

Efficiency of the induced differentiation of fibroblasts

into myofibroblasts was evaluated through the use

of positive differentiation markers, including α-SMA,

type III collagen, and ED-A FN. The IF staining for neg-

ative control markers, such as SMM and desmin, was

used to further elucidate the differentiation pathway

and exclude the induction of similar cell types.

Contributions. Conceptualization, V.D.; method-

ology, V.D.; software, Yu.L.; validation, G.S., V.D. and

P.K.; formal analysis, S.B. and P.K.; investigation, N.K.,

Yu. L. and V.D.; resources, I.E. and P.K.; data curation

P.K.; writing – original draft preparation, Yu. L.; writ-

ing – review and editing, V.D. and G.S.; visualization,

N.K., Yu.L. and V.D.; supervision, P.K.; project adminis-

tration, P.K.; funding acquisition, P.K. All authors have

read and agreed to the published version of the man-

uscript.

Funding. This work was financially supported

by the Russian Science Foundation (grant no. 23-15-

00433), https://rscf.ru/en/project/23-15-00433/.

Ethics approval and consent to participate.

This work does not contain any studies involving hu-

man and animal subjects.

Conflict of interest. The authors of this work

declare that they have no conflicts of interest.

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