Mural cell SRF controls pericyte migration, vessel patterning and blood flow

Rationale: Pericytes (PCs) and vascular smooth muscle cells (vSMCs), collectively known as mural cells(MCs), are recruited through PDGFB-PDGFRB signaling. MCs are essential for vascular integrity, and their loss has been associated with numerous diseases. Most of this knowledge is based on studies in which MCs are insufficiently recruited or fully absent upon inducible ablation. In contrast, little is known about the physiological consequences that result from impairment of specific MC functions. Objective: Here, we characterize the role of the transcription factor serum response factor (SRF) in MCs and study its function in developmental and pathological contexts. Methods and Results: We generated a mouse model of MC-specific inducible Srf gene deletion and studied its consequences during retinal angiogenesis. By postnatal day (P)6, PCs lacking SRF were morphologically abnormal and failed to properly co-migrate with angiogenic sprouts. As a consequence, PC-deficient vessels at the retinal sprouting front became dilated and leaky. By P12, also the vSMCs had lost SRF, which coincided with the formation of pathological arteriovenous (AV) shunts. Mechanistically, we show that PDGFB-dependent SRF activation is mediated via MRTF co-factors. We further show that MRTF-SRF signaling promotes pathological PC activation during ischemic retinopathy. RNA-sequencing, immunohistology, in vivo live imaging and in vitro experiments demonstrated that SRF regulates expression of contractile SMC proteins essential to maintain the vascular tone. Conclusions: SRF is crucial for distinct functions in PCs and vSMCs. SRF directs PC migration downstream of PDGFRB signaling and mediates pathological PC activation during ischemic retinopathy. In vSMCs, SRF is essential for expression of the contractile machinery, and its deletion triggers formation of AV shunts. These essential roles in physiological and pathological contexts provide a rational for novel therapeutic approaches through targeting SRF activity in MCs.

Download Full-text

Serum response factor is essential for synaptic maturation in the hippocampus

10.1101/2020.12.10.417360 ◽

2020 ◽

Author(s):

Anna Krysiak ◽

Matylda Roszkowska ◽

Lena Majchrowicz ◽

Anna Beroun ◽

Piotr Michaluk ◽

...

Keyword(s):

Dendritic Spines ◽

Serum Response Factor ◽

Imaging Techniques ◽

Expression Patterns ◽

Autism Spectrum ◽

Response Factor ◽

Serum Response

AbstractDisturbances of gene expression patterns that occur during brain development can severely affect signal transmission, connectivity, and plasticity—key features that underlie memory formation and storage in neurons. Abnormalities at the molecular level can manifest as changes in the structural and functional plasticity of dendritic spines that harbor excitatory synapses. This can lead to such developmental neuropsychiatric conditions as Autism spectrum disorders, intellectual disabilities, and schizophrenia. The present study investigated the role of the major transcriptional regulator serum response factor (SRF) in synapse maturation and its impact on behavioral phenotypes. Using in vitro and in vivo models of early postnatal SRF deletion, we studied its influence on key morphological and physiological hallmarks of spine development. The elimination of SRF in developing neurons resulted in a phenotype of immature dendritic spines and impairments in excitatory transmission. Moreover, using a combination of molecular and imaging techniques, we showed that SRF-depleted neurons exhibited a lower level of specific glutamate receptor mRNAs and a decrease in their surface expression. Additionally, the early postnatal elimination of SRF in hippocampal CA1 excitatory neurons caused spine immaturity and a specific social deficit that is frequently observed in autism patients. Altogether, our data suggest that the regulation of structural and functional dendritic spine maturation begins at the stage of gene transcription, which underpins the crucial role of such transcription factors as SRF. Moreover, disturbances of the postnatal expression of SRF translate to behavioral changes in adult animals.

Download Full-text

Serum response factor is an essential transcription factor in megakaryocytic maturation

Blood ◽

10.1182/blood-2010-01-261743 ◽

2010 ◽

Vol 116 (11) ◽

pp. 1942-1950 ◽

Cited By ~ 23

Author(s):

Stephanie Halene ◽

Yuan Gao ◽

Katherine Hahn ◽

Stephanie Massaro ◽

Joseph E. Italiano ◽

...

Keyword(s):

Transcription Factor ◽

Serum Response Factor ◽

Critical Role ◽

Response Factor ◽

Megakaryoblastic Leukemia ◽

Serum Response ◽

Essential Transcription Factor

Abstract Serum response factor (Srf) is a MADS–box transcription factor that is critical for muscle differentiation. Its function in hematopoiesis has not yet been revealed. Mkl1, a cofactor of Srf, is part of the t(1;22) translocation in acute megakaryoblastic leukemia, and plays a critical role in megakaryopoiesis. To test the role of Srf in megakaryocyte development, we crossed Pf4-Cre mice, which express Cre recombinase in cells committed to the megakaryocytic lineage, to SrfF/F mice in which functional Srf is no longer expressed after Cre-mediated excision. Pf4-Cre/SrfF/F knockout (KO) mice are born with normal Mendelian frequency, but have significant macrothrombocytopenia with approximately 50% reduction in platelet count. In contrast, the BM has increased number and percentage of CD41+ megakaryocytes (WT: 0.41% ± 0.06%; KO: 1.92% ± 0.12%) with significantly reduced ploidy. KO mice show significantly increased megakaryocyte progenitors in the BM by FACS analysis and CFU-Mk. Megakaryocytes lacking Srf have abnormal stress fiber and demarcation membrane formation, and platelets lacking Srf have abnormal actin distribution. In vitro and in vivo assays reveal platelet function defects in KO mice. Critical actin cytoskeletal genes are down-regulated in KO megakaryocytes. Thus, Srf is required for normal megakaryocyte maturation and platelet production partly because of regulation of cytoskeletal genes.

Download Full-text

Functional antagonism between YY1 and the serum response factor

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.4209-4214.1992 ◽

1992 ◽

Vol 12 (9) ◽

pp. 4209-4214

Author(s):

A Gualberto ◽

D LePage ◽

G Pons ◽

S L Mader ◽

K Park ◽

...

Keyword(s):

Serum Response Factor ◽

Regulatory Element ◽

Target Sequence ◽

Response Factor ◽

Basal Expression ◽

Actin Promoter ◽

Alpha Actin ◽

Serum Response

The rapid, transient induction of the c-fos proto-oncogene by serum growth factors is mediated by the serum response element (SRE). The SRE shares homology with the muscle regulatory element (MRE) of the skeletal alpha-actin promoter. It is not known how these elements respond to proliferative and cell-type-specific signals, but the response appears to involve the binding of the serum response factor (SRF) and other proteins. Here, we report that YY1, a multifunctional transcription factor, binds to SRE and MRE sequences in vitro. The methylation interference footprint of YY1 overlaps with that of the SRF, and YY1 competes with the SRF for binding to these DNA elements. Overexpression of YY1 repressed serum-inducible and basal expression from the c-fos promoter and repressed basal expression from the skeletal alpha-actin promoter. YY1 also repressed expression from the individual SRE and MRE sequences upstream from a TATA element. Unlike that of YY1, SRF overexpression alone did not influence the transcriptional activity of the target sequence, but SRF overexpression could reverse YY1-mediated trans repression. These data suggest that YY1 and the SRF have antagonistic functions in vivo.

Download Full-text

Cardiac Tissue Enriched Factors Serum Response Factor and GATA-4 Are Mutual Coregulators

Molecular and Cellular Biology ◽

10.1128/mcb.20.20.7550-7558.2000 ◽

2000 ◽

Vol 20 (20) ◽

pp. 7550-7558 ◽

Cited By ~ 146

Author(s):

Narasimhaswamy S. Belaguli ◽

Jorge L. Sepulveda ◽

Vishal Nigam ◽

Frédéric Charron ◽

Mona Nemer ◽

...

Keyword(s):

Zinc Finger ◽

Cardiac Tissue ◽

Serum Response Factor ◽

Zinc Finger Protein ◽

Response Factor ◽

Mads Box ◽

Cardiovascular Tissue ◽

Serum Response

ABSTRACT Combinatorial interaction among cardiac tissue-restricted enriched transcription factors may facilitate the expression of cardiac tissue-restricted genes. Here we show that the MADS box factor serum response factor (SRF) cooperates with the zinc finger protein GATA-4 to synergistically activate numerous myogenic and nonmyogenic serum response element (SRE)-dependent promoters in CV1 fibroblasts. In the absence of GATA binding sites, synergistic activation depends on binding of SRF to the proximal CArG box sequence in the cardiac and skeletal α-actin promoter. GATA-4's C-terminal activation domain is obligatory for synergistic coactivation with SRF, and its N-terminal domain and first zinc finger are inhibitory. SRF and GATA-4 physically associate both in vivo and in vitro through their MADS box and the second zinc finger domains as determined by protein A pullout assays and by in vivo one-hybrid transfection assays using Gal4 fusion proteins. Other cardiovascular tissue-restricted GATA factors, such as GATA-5 and GATA-6, were equivalent to GATA-4 in coactivating SRE-dependent targets. Thus, interaction between the MADS box and C4 zinc finger proteins, a novel regulatory paradigm, mediates activation of SRF-dependent gene expression.

Download Full-text

Three-dimensional biomimetic vascular model reveals a RhoA, Rac1, and N-cadherin balance in mural cell–endothelial cell-regulated barrier function

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1618333114 ◽

2017 ◽

Vol 114 (33) ◽

pp. 8758-8763 ◽

Cited By ~ 39

Author(s):

Stella Alimperti ◽

Teodelinda Mirabella ◽

Varnica Bajaj ◽

William Polacheck ◽

Dana M. Pirone ◽

...

Keyword(s):

Barrier Function ◽

Three Dimensional ◽

Endothelial Barrier ◽

Inflammatory Factors ◽

Mural Cell ◽

Rhoa Activity ◽

Mural Cells ◽

Adhesive Interactions

The integrity of the endothelial barrier between circulating blood and tissue is important for blood vessel function and, ultimately, for organ homeostasis. Here, we developed a vessel-on-a-chip with perfused endothelialized channels lined with human bone marrow stromal cells, which adopt a mural cell-like phenotype that recapitulates barrier function of the vasculature. In this model, barrier function is compromised upon exposure to inflammatory factors such as LPS, thrombin, and TNFα, as has been observed in vivo. Interestingly, we observed a rapid physical withdrawal of mural cells from the endothelium that was accompanied by an inhibition of endogenous Rac1 activity and increase in RhoA activity in the mural cells themselves upon inflammation. Using a system to chemically induce activity in exogenously expressed Rac1 or RhoA within minutes of stimulation, we demonstrated RhoA activation induced loss of mural cell coverage on the endothelium and reduced endothelial barrier function, and this effect was abrogated when Rac1 was simultaneously activated. We further showed that N-cadherin expression in mural cells plays a key role in barrier function, as CRISPR-mediated knockout of N-cadherin in the mural cells led to loss of barrier function, and overexpression of N-cadherin in CHO cells promoted barrier function. In summary, this bicellular model demonstrates the continuous and rapid modulation of adhesive interactions between endothelial and mural cells and its impact on vascular barrier function and highlights an in vitro platform to study the biology of perivascular–endothelial interactions.

Download Full-text

Delayed liver regeneration in mice lacking liver serum response factor

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00493.2006 ◽

2007 ◽

Vol 292 (4) ◽

pp. G996-G1001 ◽

Cited By ~ 7

Author(s):

M. Ujue Latasa ◽

Dominique Couton ◽

Claude Charvet ◽

Aurélie Lafanechère ◽

Jacques-Emmanuel Guidotti ◽

...

Keyword(s):

Liver Regeneration ◽

Partial Hepatectomy ◽

Serum Response Factor ◽

Early Response ◽

Mutant Mice ◽

Response Factor ◽

Impaired Liver ◽

Serum Response

Various immediate early genes (IEGs) upregulated during the early process of liver regeneration are transcriptional targets of the serum response factor (SRF). We show here that the expression of SRF is rapidly induced in rodent liver after partial hepatectomy. Because the inactivation of the SRF gene in mice is embryonic lethal, the in vivo role of SRF in liver regeneration after partial hepatectomy was analyzed in mutant mice conditionally deleted for SRF in the liver. We demonstrate that SRF is not an essential factor for liver ontogenesis. However, adult mutant mice show impaired liver regeneration after partial hepatectomy, associated with a blunted upregulation of various SRF target IEGs. In conclusion, our work suggests that SRF is an early response transcription factor that may contribute to the initial phases of liver regeneration through its activation of IEGs.

Download Full-text

Functional antagonism between YY1 and the serum response factor.

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.4209 ◽

1992 ◽

Vol 12 (9) ◽

pp. 4209-4214 ◽

Cited By ~ 95

Author(s):

A Gualberto ◽

D LePage ◽

G Pons ◽

S L Mader ◽

K Park ◽

...

Keyword(s):

Serum Response Factor ◽

Regulatory Element ◽

Target Sequence ◽

Response Factor ◽

Basal Expression ◽

Actin Promoter ◽

Alpha Actin ◽

Serum Response

Download Full-text

Critical Role of CIB1 in Endothelial Cell Function and In Vivo Ischemia-Induced Angiogenesis.

Blood ◽

10.1182/blood.v108.11.1800.1800 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1800-1800

Author(s):

Mohamed A. Zayed ◽

Andrew McFadden ◽

Weiping Yuan ◽

Mary E. Hartnett ◽

Dan Chalothorn ◽

...

Keyword(s):

Hind Limb ◽

Ex Vivo ◽

Critical Role ◽

Tube Formation ◽

Wild Type ◽

Wild Type Littermate ◽

Retinal Angiogenesis

Abstract CIB1, a 22kDa EF-hand containing calcium binding protein, was originally identified in a yeast two-hybrid screen as a binding partner for the cytoplasmic tail of the platelet integrin αIIb. CIB1 also associates with a number of kinases and modulates their activity, suggesting that CIB1 is an important regulatory molecule. Recently, we found that CIB1 is expressed in multiple endothelial cell (EC) types. We therefore tested the role of CIB1 in EC function in vitro, and in angiogenesis both ex vivo and in vivo. To test the role of CIB1 in EC function in vitro, we reduced endogenous CIB1 levels in ECs by RNA interference with an shRNA-delivered by lentivirus. CIB1 depletion significantly decreased EC haptotaxis on fibronectin and EC vascular tube formation on growth factor-reduced Matrigel. Treatment with FGF-2, an angiogenic factor, did not counter the observed inhibition of haptotaxis and tube formation by shRNA against CIB1. However, CIB1 overexpression enhanced FGF-2-induced EC haptotaxis relative to control cells. Similarly, ECs derived from CIB1 null mice exhibited a significant decrease in haptotaxis, tube formation, and proliferation compared to ECs isolated from wild-type littermate controls. In ex vivo aortic ring and tibialis anterior muscle culture assays, CIB1 null cultures supplemented with serum or FGF-2 demonstrated reduced blood vessel sprouting compared to wild-type littermate control cultures. Finally, in vivo assays for hyperoxic retinal angiogenesis and hind-limb induced-ischemia revealed a decrease in post-ischemia retinal neovascularization and Doppler hind-limb blood perfusion recovery, although developmental retinal angiogenesis in CIB1 null mice appeared normal. In conclusion, these findings support a critical role for CIB1 in EC function that appears to be important for ischemia-induced angiogenesis.

Download Full-text

Inhibition of Serum Response Factor Improves Response to Enzalutamide in Prostate Cancer

Cancers ◽

10.3390/cancers12123540 ◽

2020 ◽

Vol 12 (12) ◽

pp. 3540

Author(s):

R. William Watson ◽

Haleema Azam ◽

Claudia Aura ◽

Niamh Russell ◽

Janet McCormack ◽

...

Keyword(s):

Prostate Cancer ◽

Serum Response Factor ◽

Specific Antigen ◽

Xenograft Model ◽

Tumour Volume ◽

Lead Target ◽

Response Factor ◽

Serum Response

Castrate-resistant prostate cancer (CRPC) is challenging to treat with the androgen receptor (AR), the main target and key focus of resistance. Understanding the mechanisms of AR interaction with co-regulators will identify new therapeutic targets to overcome AR resistance mechanisms. We previously identified the serum response factor (SRF) as a lead target in an in vitro model of CRPC and showed that SRF expression in tissues of CRPC patients was associated with shorter survival. Here, we tested SRF inhibition in vitro and in vivo to assess SRF as a potential target in CRPC. Inhibition of SRF with the small-molecule inhibitor CCG1423 resulted in enhanced response to enzalutamide in vitro and reduced tumour volume of LuCaP 35CR, a CRPC patient-derived xenograft model. Nuclear localisation of AR post-CCG1423 was significantly decreased and was associated with decreased α-tubulin acetylation in vitro and decreased prostate specific antigen (PSA) levels in vivo. SRF immunoreactivity was tested in metastatic tissues from CRPC patients to investigate its role in enzalutamide response. Kaplan–Meier curves showed that high SRF expression was associated with shorter response to enzalutamide. Our study supports the use of SRF inhibitors to improve response to enzalutamide.

Download Full-text

Protein Kinase Cδ Blocks Immediate-Early Gene Expression in Senescent Cells by Inactivating Serum Response Factor

Molecular and Cellular Biology ◽

10.1128/mcb.24.16.7298-7311.2004 ◽

2004 ◽

Vol 24 (16) ◽

pp. 7298-7311 ◽

Cited By ~ 25

Author(s):

Keith Wheaton ◽

Karl Riabowol

Keyword(s):

Gene Expression ◽

Protein Kinase ◽

Serum Response Factor ◽

Immediate Early Gene ◽

Early Gene ◽

Immediate Early ◽

Response Factor ◽

Serum Response

ABSTRACT Fibroblasts lose the ability to replicate in response to growth factors and become unable to express growth-associated immediate-early genes, including c-fos and egr-1, as they become senescent. The serum response factor (SRF), a major transcriptional activator of immediate-early gene promoters, loses the ability to bind to the serum response element (SRE) and becomes hyperphosphorylated in senescent cells. We identify protein kinase C delta (PKCδ) as the kinase responsible for inactivation of SRF both in vitro and endogenously in senescent cells. This is due to a higher level of PKCδ activity as cells age, production of the PKCδ catalytic fragment, and its nuclear localization in senescent but not in low-passage-number cells. The phosphorylation of T160 of SRF by PKCδ in vitro and in vivo led to loss of SRF DNA binding activity. Both the PKCδ inhibitor rottlerin and ectopic expression of a dominant negative form of PKCδ independently restored SRE-dependent transcription and immediate-early gene expression in senescent cells. Modulation of PKCδ activity in vivo with rottlerin or bistratene A altered senescent- and young-cell morphology, respectively. These observations support the idea that the coordinate transcriptional inhibition of several growth-associated genes by PKCδ contributes to the senescent phenotype.

Download Full-text