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Purpose: Intentional or accidental exposure of relatively large as well as localized areas of the skin to ionizing radiation can lead to severe damage of many of its cellular components and cutaneous radiation syndrome. Patients can be treated with an invasive surgical procedure coupled with autologous cell therapy. However, this approach remains perfectible, especially for muscle repair. Indeed, a severe underlying muscle defect persists, in particular because of the damage to the satellite cells which ensure muscle regeneration. To overcome these shortcomings, a solution could be to develop new therapeutic strategies based on pharmacological treatments to improve post-irradiation muscle regeneration. In this study, we focus on the Hedgehog signaling pathway as a target, due to its involvement in myogenesis. Materials and methods: To evaluate the benefit of the pro-myogenic Hedgehog signaling pathway modulation, recombinant Sonic Hedgehog (rShh; agonist) or Cyclopamine (antagonist) were used in a stable cell line of mouse C2C12 myoblasts exposed to radiation (X-rays; 5 Gy). Our in vitro studies were carried out under either proliferation or differentiation conditions. Proliferation, migration, survival (apoptosis) and expression of myogenic genes/proteins were evaluated. Results: A high dose of radiation was shown to exert a serious negative impact in our in vitro model of mouse muscle progenitors after irradiation in proliferation or differentiation conditions. Interestingly, Hh pathway stimulation by rShh promotes the proliferation of myoblasts and their survival while its blockade by Cyclopamine significantly increases cell differentiation toward mature myotubes. Conclusion: These data suggest that, after irradiation, the sequence of activation and inhibition of the Hh pathway could allow rescue and proliferation of satellite cells, followed by their differentiation to regenerate new fibers. On the basis of these encouraging in vitro results, the second phase of our study will involve the in vivo validation of this treatment in a new murine model of ultra-localized muscle irradiation. [ABSTRACT FROM AUTHOR]

Deficient Anterior pituitary with common Variable Immune Deficiency (DAVID) syndrome results from NFKB2 heterozygous mutations, causing adrenocorticotropic hormone deficiency (ACTHD) and primary hypogammaglobulinemia. While NFKB signaling plays a crucial role in the immune system, its connection to endocrine symptoms is unclear. We established a human disease model to investigate the role of NFKB2 in pituitary development by creating pituitary organoids from CRISPR/Cas9-edited human induced pluripotent stem cells (hiPSCs). Introducing homozygous TBX19 K146R/K146R missense pathogenic variant in hiPSC, an allele found in congenital isolated ACTHD, led to a strong reduction of corticotrophs number in pituitary organoids. Then, we characterized the development of organoids harboring NFKB2 D865G/D865G mutations found in DAVID patients. NFKB2 D865G/D865G mutation acted at different levels of development with mutant organoids displaying changes in the expression of genes involved on pituitary progenitor generation ( HESX1 , PITX1 , LHX3 ), hypothalamic secreted factors ( BMP4, FGF8, FGF10 ), epithelial-to-mesenchymal transition, lineage precursors development ( TBX19 , POU1F1 ) and corticotrophs terminal differentiation ( PCSK1, POMC ), and showed drastic reduction in the number of corticotrophs. Our results provide strong evidence for the direct role of NFKB2 mutations in the endocrine phenotype observed in patients leading to a new classification of a NFKB2 variant of previously unknown clinical significance as pathogenic in pituitary development., Competing Interests: TM, TF, NJ, PR, HE, AB, FC, TB No competing interests declared, (© 2023, Mac et al.)

Clathrin-mediated endocytosis has characteristic features in neuronal dendrites and presynapses, but how membrane proteins are internalized along the axon shaft remains unclear. We focused on clathrin-coated structures and endocytosis along the axon initial segment (AIS) and their relationship to the periodic actin-spectrin scaffold that lines the axonal plasma membrane. A combination of super-resolution microscopy and platinum-replica electron microscopy on cultured neurons revealed that AIS clathrin-coated pits form within "clearings", circular areas devoid of actin-spectrin mesh. Actin-spectrin scaffold disorganization increased clathrin-coated pit formation. Cargo uptake and live-cell imaging showed that AIS clathrin-coated pits are particularly stable. Neuronal plasticity-inducing stimulation triggered internalization of the clathrin-coated pits through polymerization of branched actin around them. Thus, spectrin and actin regulate clathrin-coated pit formation and scission to control endocytosis at the AIS.

The current geopolitical context has brought the radiological nuclear risk to the forefront of concerns. High-dose localized radiation exposure leads to the development of a musculocutaneous radiation syndrome affecting the skin and subcutaneous muscles. Despite the implementation of a gold standard treatment based on an invasive surgical procedure coupled with autologous cell therapy, a muscular defect frequently persists. Targeting the modulation of the Hedgehog (Hh) signaling pathway appears to be a promising therapeutic approach. Activation of this pathway enhances cell survival and promotes proliferation after irradiation, while inhibition by Cyclopamine facilitates differentiation. In this study, we compared the effects of three antagonists of Hh, Cyclopamine (CA), Vismodegib (VDG) and Sonidegib (SDG) on differentiation. A stable cell line of murine myoblasts, C2C12, was exposed to X-ray radiation (5 Gy) and treated with CA, VDG or SDG. Analysis of proliferation, survival (apoptosis), morphology, myogenesis genes expression and proteins production were performed. According to the results, VDG does not have a significant impact on C2C12 cells. SDG increases the expression/production of differentiation markers to a similar extent as CA, while morphologically, SDG proves to be more effective than CA. To conclude, SDG can be used in the same way as CA but already has a marketing authorization with an indication against basal cell cancers, facilitating their use in vivo. This proof of concept demonstrates that SDG represents a promising alternative to CA to promotes differentiation of murine myoblasts. Future studies on isolated and cultured satellite cells and in vivo will test this proof of concept., (©2024 by Radiation Research Society. All rights of reproduction in any form reserved.)

The architecture of the actin cytoskeleton that concentrates at presynapses remains poorly known, hindering our understanding of its roles in synaptic physiology. In this work, we measure and visualize presynaptic actin by diffraction-limited and super-resolution microscopy, thanks to a validated model of bead-induced presynapses in cultured neurons. We identify a major population of actin-enriched presynapses that concentrates more presynaptic components and shows higher synaptic vesicle cycling than their non-enriched counterparts. Pharmacological perturbations point to an optimal actin amount and the presence of distinct actin structures within presynapses. We directly visualize these nanostructures using Single Molecule Localization Microscopy (SMLM), defining three distinct types: an actin mesh at the active zone, actin rails between the active zone and deeper reserve pools, and actin corrals around the whole presynaptic compartment. Finally, CRISPR-tagging of endogenous actin allows us to validate our results in natural synapses between cultured neurons, confirming the role of actin enrichment and the presence of three types of presynaptic actin nanostructures., (© 2023 Bingham et al.)

Deficient anterior pituitary with common variable immune deficiency (DAVID) syndrome is a rare condition characterized by adrenocorticotropic hormone (ACTH) deficiency and primary hypogammaglobulinemia. It is due to heterozygous mutations of the nuclear factor kappa-B subunit 2 (NFKB2) gene. Only a few isolated cases have been reported since its first description by our team. Through the international multicenter GENHYPOPIT network, we identified a new case of DAVID syndrome. We then conducted an extensive review of the DAVID syndrome cases published from 2012 to 2022. A 7-year-old boy was diagnosed with symptomatic hypoglycemia revealing ACTH deficiency. Laboratory tests showed asymptomatic hypogammaglobulinemia. He harbored a heterozygous point mutation in NFKB2 gene (c.2600C > T, p.Ala867Val). His management included hydrocortisone replacement treatment, and he also received subcutaneous immunoglobulins during the Covid-19 pandemic. We analyzed 28 cases of DAVID syndrome with ACTH deficiency. ACTH deficiency was the only hormone deficiency in 79% of patients, but some patients harbored growth hormone (GH) and thyroid stimulating hormone (TSH) deficiencies. The first presenting symptoms were sinus/pulmonary infections (82%, mean age of 3 years) and alopecia (mean age of 4.7 years). ACTH deficiency was the third presenting condition (mean age at diagnosis of 8.6 years). All patients had hypogammaglobulinemia (decreased IgA and IgM levels), and 57% of patients had at least one autoimmune manifestation. Heterozygous mutations at the 3'end of the NFKB2 gene, coding for the C-terminal domain of the protein, were identified in all cases. Better knowledge of DAVID syndrome will help clinicians make an early diagnosis to avoid life-threatening complications., (© 2023 British Society for Neuroendocrinology.)

Apolipoprotein E4 (APOEε4) is the major allelic risk factor for late-onset sporadic Alzheimer's disease (sAD). Inflammation is increasingly considered as critical in sAD initiation and progression. Identifying brain molecular mechanisms that could bridge these two risk factors remain unelucidated. Leveraging induced pluripotent stem cell (iPSC)-based strategies, we demonstrate that APOE controls inflammation in human astrocytes by regulating Transgelin 3 (TAGLN3) expression and, ultimately, nuclear factor κB (NF-κB) activation. We uncover that APOE4 specifically downregulates TAGLN3, involving histone deacetylases activity, which results in low-grade chronic inflammation and hyperactivated inflammatory responses. We show that APOE4 exerts a dominant negative effect to prime astrocytes toward a pro-inflammatory state that is pharmacologically reversible by TAGLN3 supplementation. We further confirm that TAGLN3 is downregulated in the brain of patients with sAD. Our findings highlight the APOE-TAGLN3-NF-κB axis regulating neuroinflammation in human astrocytes and reveal TAGLN3 as a molecular target to modulate neuroinflammation, as well as a potential biomarker for AD., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The widespread availability of recombineered vectors and gene targeted embryonic stem cells from large-scale repositories facilitates the generation of mouse models for functional genetic studies. Southern blotting validates the structure of these targeted alleles produced by homologous recombination, as well as indicating any additional integrations of the vector into the genome. Traditionally this technique employs radioactively-labelled probes; however, there are many laboratories that are restricted in their use of radioactivity. Here, we present a widely applicable protocol for Southern blot analysis using cold probes and alternative procedures employing radioactive probes. Furthermore, the probes are designed to recognise standardised regions of gene-targeting cassettes and so represent universally applicable reagents for assessing allelic integrity., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

We previously discovered the implication of membrane-type 5-matrix metalloproteinase (MT5-MMP) in Alzheimer's disease (AD) pathogenesis. Here, we shed new light on pathogenic mechanisms by which MT5-MMP controls the processing of amyloid precursor protein (APP) and the fate of amyloid beta peptide (Aβ) as well as its precursor C99, and C83. We found in human embryonic kidney cells (HEK) carrying the APP Swedish familial mutation (HEKswe) that deleting the C-terminal non-catalytic domains of MT5-MMP hampered its ability to process APP and release the soluble 95 kDa form (sAPP95). Catalytically inactive MT5-MMP variants increased the levels of Aβ and promoted APP/C99 sorting in the endolysosomal system, likely through interactions of the proteinase C-terminal portion with C99. Most interestingly, the deletion of the C-terminal domain of MT5-MMP caused a strong degradation of C99 by the proteasome and prevented Aβ accumulation. These discoveries reveal new control of MT5-MMP over APP by proteolytic and non-proteolytic mechanisms driven by the C-terminal domains of the proteinase. The targeting of these non-catalytic domains of MT5-MMP could, therefore, provide new insights into the therapeutic regulation of APP-related pathology in AD., (© 2021 Federation of American Societies for Experimental Biology.)

Autism spectrum disorders (ASD) are complex neurodevelopmental disorders with a very large number of risk loci detected in the genome. However, at best, each of them explains rare cases, the majority being idiopathic. Genomic data on ASD derive mostly from post-mortem brain analyses or cell lines derived from blood or patient-specific induced pluripotent stem cells (iPSCS). Therefore, the transcriptional and regulatory architecture of the nervous system, particularly during early developmental periods, remains highly incomplete. To access the critical disturbances that may have occurred during pregnancy or early childhood, we recently isolated stem cells from the nasal cavity of anesthetized patients diagnosed for ASD and compared them to stem cells from gender-matched control individuals without neuropsychiatric disorders. This allowed us to discover MOCOS, a non-mutated molybdenum cofactor sulfurase-coding gene that was under-expressed in the stem cells of most ASD patients of our cohort, disturbing redox homeostasis and synaptogenesis. We now report that a divergent transcription upstream of MOCOS generates an antisense long noncoding RNA, to which we coined the name COSMOC. Surprisingly, COSMOC is strongly under-expressed in all ASD patients of our cohort with the exception of a patient affected by Asperger syndrome. Knockdown studies indicate that loss of COSMOC reduces MOCOS expression, destabilizes lipid and energy metabolisms of stem cells, but also affects neuronal maturation and splicing of synaptic genes. Impaired expression of the COSMOC/MOCOS bidirectional unit might shed new lights on the origins of ASD that could be of importance for future translational studies.

Context: The international GENHYPOPIT network collects phenotypical data and screens genetic causes of non-acquired hypopituitarism., Aims: To describe main phenotype patterns and their evolution through life., Design: Patients were screened according to their phenotype for coding sequence variations in 8 genes: HESX1, LHX3, LHX4, PROP1, POU1F1, TBX19, OTX2 and PROKR2., Results: Among 1213 patients (1143 index cases), the age of diagnosis of hypopituitarism was congenital (24%), in childhood (28%), at puberty (32%), in adulthood (7.2%) or not available (8.8%). Noteworthy, pituitary hormonal deficiencies kept on evolving during adulthood in 49 of patients. Growth Hormone deficiency (GHD) affected 85.8% of patients and was often the first diagnosed deficiency. AdrenoCorticoTropic Hormone deficiency rarely preceded GHD, but usually followed it by over 10 years. Pituitary Magnetic Resonance Imaging (MRI) abnormalities were common (79.7%), with 39.4% pituitary stalk interruption syndrome (PSIS). The most frequently associated extrapituitary malformations were ophthalmological abnormalities (16.1%). Prevalence of identified mutations was 7.3% of index cases (84/1143) and 29.5% in familial cases (n = 146). Genetic analysis in 449 patients without extrapituitary phenotype revealed 36 PROP1, 2 POU1F1 and 17 TBX19 mutations., Conclusion: This large international cohort highlights atypical phenotypic presentation of constitutional hypopituitarism, such as post pubertal presentation or adult progression of hormonal deficiencies. These results justify long-term follow-up, and the need for systematic evaluation of associated abnormalities. Genetic defects were rarely identified, mainly PROP1 mutations in pure endocrine phenotypes., (© 2020 John Wiley & Sons Ltd.)

LHX3 is an LIM domain transcription factor involved in the early steps of pituitary ontogenesis. We report here functional studies of three allelic variants, including the first heterozygous variant of LHX3 NM_178138.5(LHX3):c.587T>C (p.(Leu196Pro)) that may be responsible for a milder phenotype of hypopituitarism. Our functional studies showed that NM_178138.5(LHX3):c.587T>C (p.(Leu196Pro)) was not able to activate target promoters in vitro, as it did not bind DNA, and likely affected LHX3 function via a mechanism of haplo-insufficiency. Our study demonstrates the possibility that patients with a heterozygous variant of LHX3 may have pituitary deficiencies, with a milder phenotype than patients with homozygous variants. It is thus of vital to propose an optimal follow-up of such patients, who, until now, were considered as not being at risk of presenting pituitary deficiency. The second variant NM_178138.5(LHX3):c.622C>G (p.(Arg208Gly)), present in a homozygous state, displayed decreased transactivating ability without loss of binding capacity in vitro, concordant with in silico analysis; it should thus be considered to affect LHX3 function. In contrast, the NM_178138.5(LHX3):c.929G>C (p.(Arg310Pro)) variant, in a heterozygous state, also predicted as deleterious in silico, proved functionally active in vitro, and should thus still be classified as a variant of unknown significance. Our study emphasizes the need for functional studies due to the limits of software-based predictions of new variants, and the possible association of a pituitary phenotype to heterozygous LHX3 variants.

Purpose: Degenerative disc disease involves sequential events that lead to the loss of cells, a decrease in disc matrix production, disc dehydration, and alteration of its biomechanical properties. The aim of this study was to determine whether cryoinjury of the nucleus pulposus performed through endplate perforation contributes to disc degeneration and to compare this technique with standard methods., Method: Under general anesthesia, the lumbar discs of six pigs were exposed and randomly submitted to needle puncture of the annulus fibrosus (NeP), isolated endplate injury (EP), or cryoinjury using a 2.5-J Thompson cryoprobe applied through a single endplate perforation (EP+cryo). The remaining discs served as controls. Animals were sacrificed at two months and the harvested lumbar spines were submitted to CT scan and MRI investigations. Histologic analysis was performed to assess the degree of disc degeneration., Results: CT scan showed that decrease in average disc height was more important after cryoinjury (49.3%) than after endplate perforation (16.9%) (P < 0.0001) or needle puncture (19.4%) (P < 0.0001). On MRI, the dehydration ratio was significantly more important after EP+cryo (60%) than after NP (40%) or EP (30%) (P < 0.0001). After cryoinjury, the histologic score developed for this study was significantly higher than after needle puncture or endplate perforation (P < 0.0001)., Conclusions: Imaging and histological analysis showed that disc cryoinjury applied through endplate perforation was superior to the classical NeP and EP models to induce experimental disc degeneration. This model appears suitable for testing safety and efficacy of novel treatments of intervertebral disc degeneration.

The cutaneous radiation syndrome is the clinical consequence of local high-dose irradiation. It is characterized by extensive inflammation, necrosis, and poor revascularization of the skin, resulting in muscle inflammation and fibrosis. Based on these physiopathological processes, subcutaneous injections of adipose-tissue-derived stem/stromal cells have shown favorable effects on skin-wound healing in a minipig model of cutaneous radiation syndrome, in which muscle fibrosis persisted. Since fibrosis is mainly due to the inflammatory processes that often affect underlying tissues as well, the beneficial effects of intramuscular injections of adipose-tissue-derived stem/stromal cells on tissue recovery were evaluated. The polarization of the inflammatory response of irradiated muscle in a minipig model of cutaneous radiation syndrome was determined after acute local irradiation with 50 Gy gamma rays in a preliminary study (six minipigs). Analysis of the main inflammatory cytokines of the inflammatory response M1 (IL-1-beta and IL-6) and M2 (IL-10 and TGF-beta) by western blotting and in situ hybridization, as well as analysis of CD80/CD206 M1/M2 macrophage-specific markers by immunohistochemistry on minipig muscle samples, was performed 76 d after irradiation. The treatment of irradiated muscles with autologous adipose-tissue-derived stem/stromal cells led to an increase in IL-10 and TGF-beta, being associated with an increase in CD68+/CD206+ cells in this area. This highlights a polarization of M2 in the inflammatory response and indicates that adipose-tissue-derived stem/stromal cells may direct the irradiated tissues' inflammatory response towards a proregenerative outcome.

Through the multicentric international GENHYPOPIT network, 10 transcription factor genes involved in pituitary development have been screened in more than 1200 patients with constitutional hypopituitarism over the past two decades. The present report summarizes the main lessons learned from this phenotype-based genetic screening: (1) genetically determined hypopituitarism does not necessarily present during childhood; (2) constitutional hypopituitarism may be characterized by a pure endocrine phenotype or by various combinations of endocrine deficits and visceral malformations; (3) syndromic hypopituitarism may also be observed in patients with POU1F1 or PROP1 mutations; (4) in cases of idiopathic hypopituitarism, extensive genetic screening identifies gene alterations in a minority of patients; (5) functional studies are imperfect in determining the involvement of an allelic variant in a specific pituitary phenotype., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Background/aim: Congenital hypothyroidism (CH) is a heterogeneous entity. Neonatal screening programs based on thyrotropin (TSH) determination allow primary CH diagnosis but miss central CH (CCH). CCH causes morbidity, alerts to other pituitary deficiencies, and is more prevalent than previously thought. We aimed at developing a pilot neonatal screening program for CCH detection., Patients and Methods: A prospective 2-year pilot neonatal screening study based on simultaneous dried blood specimen TSH and thyroxine (T4) measurements was implemented in term newborns aged 2-7 days. Those with T4 ≤4.5 µg/dL (-2.3 SDS) and TSH <10 mIU/L were recalled (suspicious of CCH) and underwent clinical and biochemical assessment performed by expert pediatric endocrinologists., Results: A total of 67,719 newborns were screened. Primary CH was confirmed in 24 (1: 2,821). Forty-four newborns with potential CCH were recalled (recall rate 0.07%) at a mean age of 12.6 ± 4.8 days. In this group, permanent CCH was confirmed in 3 (1: 22,573), starting L-T4 treatment at a mean age of 12.3 ± 6.6 days; 14 boys showed T4-binding globulin deficiency (1: 4,837); 24 had transient hypothyroxinemia (21 non-thyroidal illness and 3 healthy); and 3 died before the confirmation stage. According to initial free T4 measurements, CCH patients had moderate hypothyroidism., Conclusions: Adding T4 to TSH measurements enabled the identification of CCH as a prevalent condition and contributed to improving the care of newborns with congenital hypopituitarism and recognizing other thyroidal disorders., (© 2017 S. Karger AG, Basel.)

Over the last 5 years, new actors involved in the pathogenesis of combined pituitary hormone deficiency in humans have been reported: they included a member of the immunoglobulin superfamily glycoprotein and ciliary G protein-coupled receptors, as well as new transcription factors and signalling molecules. New modes of inheritance for alterations of genes encoding transcription factors have also been described. Finally, actors known to be involved in a very specific phenotype (hypogonadotroph hypogonadism for instance) have been identified in a wider range of phenotypes. These data thus suggest that new mechanisms could explain the low rate of aetiological identification in this heterogeneous group of diseases. Taking into account the fact that several reviews have been published in recent years on classical aetiologies of CPHD such as mutations of POU1F1 or PROP1, we focused the present overview on the data published in the last 5 years, to provide the reader with an updated review on this rapidly evolving field of knowledge., (© 2016 European Society of Endocrinology.)

Osteonecrosis of the femoral head is a frequent complication in adult patients with sickle cell disease (SCD). To delay hip arthroplasty, core decompression combined with concentrated total bone marrow (BM) treatment is currently performed in the early stages of the osteonecrosis. Cell therapy efficacy depends on the quantity of implanted BM stromal cells. For this reason, expanded bone marrow stromal cells (BMSCs, also known as bone marrow derived mesenchymal stem cells) can be used to improve osteonecrosis treatment in SCD patients. In this study, we quantitatively and qualitatively evaluated the function of BMSCs isolated from a large number of SCD patients with osteonecrosis (SCD-ON) compared with control groups (patients with osteonecrosis not related to SCD (ON) and normal donors (N)). BM total nuclear cells and colony-forming efficiency values (CFE) were significantly higher in SCD-ON patients than in age and sex-matched controls. The BMSCs from SCD-ON patients were similar to BMSCs from the control groups in terms of their phenotypic and functional properties. SCD-ON patients have a higher frequency of BMSCs that retain their bone regeneration potential. Our findings suggest that BMSCs isolated from SCD-ON patients can be used clinically in cell therapy approaches. This work provides important preclinical data that is necessary for the clinical application of expanded BMSCs in advanced therapies and medical products.

LHX4 is a LIM homeodomain transcription factor involved in the early steps of pituitary ontogenesis. To date, 8 heterozygous LHX4 mutations have been reported as responsible of combined pituitary hormone deficiency (CPHD) in Humans. We identified 4 new LHX4 heterozygous allelic variants in patients with congenital hypopituitarism: W204X, delK242, N271S and Q346R. Our objective was to determine the role of LHX4 variants in patients' phenotypes. Heterologous HEK293T cells were transfected with plasmids encoding for wild-type or mutant LHX4. Protein expression was analysed by Western Blot, and DNA binding by electro-mobility shift assay experiments. Target promoters of LHX4 were cotransfected with wild type or mutant LHX4 to test the transactivating abilities of each variant. Our results show that the W204X mutation was associated with early GH and TSH deficiencies and later onset ACTH deficiency. It led to a truncated protein unable to bind to alpha-Gsu promoter binding consensus sequence. W204X was not able to activate target promoters in vitro. Cotransfection experiments did not favour a dominant negative effect. In contrast, all other mutants were able to bind the promoters and led to an activation similar as that observed with wild type LHX4, suggesting that they were likely polymorphisms. To conclude, our study underlines the need for functional in vitro studies to ascertain the role of rare allelic variants of LHX4 in disease phenotypes. It supports the causative role of the W204X mutation in CPHD and adds up childhood onset ACTH deficiency to the clinical spectrum of the various phenotypes related to LHX4 mutations.

To test the role of wtPIT-1 (PITWT) or PIT-1 (R271W) (PIT271) in somatolactotroph cells, we established, using inducible lentiviral vectors, sublines of GH4C1 somatotroph cells that allow the blockade of the expression of endogenous PIT-1 and/or the expression of PITWT or PIT271, a dominant negative mutant of PIT-1 responsible for Combined Pituitary Hormone Deficiency in patients. Blocking expression of endogenous PIT-1 induced a marked decrease of cell proliferation. Overexpressing PITWT twofold led also to a dose-dependent decrease of cell proliferation that was accompanied by cell death. Expression of PIT271 induced a strong dose-dependent decrease of cell proliferation accompanied by a very pronounced cell death. These actions of PIT271 are independent of its interaction/competition with endogenous PIT-1, as they were unchanged when expression of endogenous PIT-1 was blocked. All these actions are specific for somatolactotroph cells, and could not be observed in heterologous cells. Cell death induced by PITWT or by PIT271 was accompanied by DNA fragmentation, but was not inhibited by inhibitors of caspases, autophagy or necrosis, suggesting that this cell death is a caspase-independent apoptosis. Altogether, our results indicate that under normal conditions PIT-1 is important for the maintenance of cell proliferation, while when expressed at supra-normal levels it induces cell death. Through this dual action, PIT-1 may play a role in the expansion/regression cycles of pituitary lactotroph population during and after lactation. Our results also demonstrate that the so-called "dominant-negative" action of PIT271 is independent of its competition with PIT-1 or a blockade of the actions of the latter, and are actions specific to this mutant variant of PIT-1.