Tendon Extracellular Matrix Alterations in Ullrich Congenital Muscular Dystrophy.

Collagen VI (COLVI) is a non-fibrillar collagen expressed in skeletal muscle and most connective tissues. Mutations in COLVI genes cause two major clinical forms, Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD). In addition to congenital muscle weakness, patients affected by COLVI myopathies show axial and proximal joint contractures and distal joint hypermobility, which suggest the involvement of the tendon function. We examined a peroneal tendon biopsy and tenocyte culture of a 15-year-old patient affected by UCMD with compound heterozygous COL6A2 mutations. In patient's tendon biopsy, we found striking morphological alterations of tendon fibrils, consisting in irregular profiles and reduced mean diameter. The organization of the pericellular matrix of tenocytes, the primary site of collagen fibril assembly, was severely affected, as determined by immunoelectron microscopy, which showed an abnormal accumulation of COLVI and altered distribution of collagen I (COLI) and fibronectin (FBN). In patient's tenocyte culture, COLVI web formation and cell surface association were severely impaired; large aggregates of COLVI, which matched with COLI labeling, were frequently detected in the extracellular matrix. In addition, metalloproteinase MMP-2, an extracellular matrix-regulating enzyme, was increased in the conditioned medium of patient's tenocytes, as determined by gelatin zymography and western blot. Altogether, these data indicate that COLVI deficiency may influence the organization of UCMD tendon matrix, resulting in dysfunctional fibrillogenesis. The alterations of tendon matrix may contribute to the complex pathogenesis of COLVI related myopathies.

Collagen VI-NG2 axis in human tendon fibroblasts under conditions mimicking injury response.

In response to injury, tendon fibroblasts are activated, migrate to the wound, and contribute to tissue repair by producing and organizing the extracellular matrix. Collagen VI is a microfibrillar collagen enriched in the pericellular matrix of tendon fibroblasts with a potential regulatory role in tendon repair mechanism. We investigated the molecular basis of the interaction between collagen VI and the cell membrane both in tissue sections and fibroblast cultures of human tendon, and analyzed the deposition of collagen VI during migration and myofibroblast trans-differentiation, two crucial events for tendon repair. Tendon fibroblast displayed a collagen VI microfibrillar network closely associated with the cell surface. Binding of collagen VI with the cell membrane was mediated by NG2 proteoglycan, as demonstrated by in vitro perturbation of collagen VI-NG2 interaction with a NG2-blocking antibody. Cultures subjected to wound healing scratch assay displayed collagen VI-NG2 complexes at the trailing edge of migrating cells, suggesting a potential role in cell migration. In fact, the addition of a NG2-blocking antibody led to an impairment of cell polarization and delay of wound closure. Similar results were obtained after in vitro perturbation of collagen VI extracellular assembly with the 3C4 anti-collagen VI antibody and in collagen VI-deficient tendon cultures of a Ullrich congenital muscular dystrophy patient carrying mutations in COL6A2 gene. Moreover, in vitro treatment with transforming growth factor ß1 (TGFß1) induced a dramatic reduction of NG2 expression, both at protein and mRNA transcript level, and the impairment of collagen VI association with the cell membrane. Instead, collagen VI was still detectable in the extracellular matrix in association with ED-A fibronectin and collagen I, which were strongly induced by TGFß1 treatment. Our findings reveal a critical role of the NG2 proteoglycan for the binding of collagen VI to the surface of tendon fibroblasts. By interacting with NG2 proteoglycan and other extracellular matrix proteins, collagen VI regulates fibroblasts behavior and the assembly of tendon matrix, thereby playing a crucial role in tendon repair.

Assessment of an efficient xeno-free culture system of human periodontal ligament stem cells.

The possibility of transplanting adult stem cells into damaged organs has opened new prospects for the treatment of several human pathologies. The purpose of this study was to develop a culture system for the expansion and production of human Periodontal Ligament Stem Cells (hPDLSCs) using a new xeno-free media formulation and ensuring the maintenance of the stem cells features comprising the multiple passage expansion, mesengenic lineage differentiation, cellular phenotype, and genomic stability, essential elements for conforming to translation to cell therapy. Somatic stem cells were isolated from the human periodontium using a minimally invasive periodontal access flap surgery in healthy donors. Expanded hPDLSCs in a xeno-free culture showed the morphological features of stem cells, expressed the markers associated with pluripotency, and a normal karyotype. Under appropriate culture conditions, hPDLSCs presented adipogenic and osteogenic potential; indeed, a very high accumulation of lipid droplets was evident in the cytoplasm of adipogenic-induced cells, and indisputable evidence of osteogenic differentiation, investigated by transmission electron microscopy, and analyzed for gene expression analysis has been shown. Based on these data, the novel xeno-free culture method might provide the basis for Good Manufacturing Procedure culture of autologous stem cells, readily accessible from human periodontium, and can be a resource to facilitate their use in human clinical studies for potential therapeutic regeneration.

Melanocytes from Patients Affected by Ullrich Congenital Muscular Dystrophy and Bethlem Myopathy have Dysfunctional Mitochondria That Can be Rescued with Cyclophilin Inhibitors.

Ullrich congenital muscular dystrophy and Bethlem myopathy are caused by mutations in collagen VI (ColVI) genes, which encode an extracellular matrix protein; yet, mitochondria play a major role in disease pathogenesis through a short circuit caused by inappropriate opening of the permeability transition pore, a high-conductance channel, which causes a shortage in ATP production. We find that melanocytes do not produce ColVI yet they bind it at the cell surface, suggesting that this protein may play a trophic role and that its absence may cause lesions similar to those seen in skeletal muscle. We show that mitochondria in melanocytes of Ullrich congenital muscular dystrophy and Bethlem myopathy patients display increased size, reduced matrix density, and disrupted cristae, findings that suggest a functional impairment. In keeping with this hypothesis, mitochondria (i) underwent anomalous depolarization after inhibition of the F-ATP synthase with oligomycin, and (ii) displayed decreased respiratory reserve capacity. The non-immunosuppressive cyclophilin inhibitor NIM811 prevented mitochondrial depolarization in response to oligomycin in melanocytes from both Ullrich congenital muscular dystrophy and Bethlem myopathy patients, and partially restored the respiratory reserve of melanocytes from one Bethlem myopathy patient. These results match our recent findings on melanocytes from patients affected by Duchenne muscular dystrophy (Pellegrini et al., 2013), and suggest that skin biopsies may represent a minimally invasive tool to investigate mitochondrial dysfunction and to evaluate drug efficacy in ColVI-related myopathies and possibly in other muscle wasting conditions like aging sarcopenia.

NIM811, a cyclophilin inhibitor without immunosuppressive activity, is beneficial in collagen VI congenital muscular dystrophy models.

Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) are inherited muscle diseases due to mutations in the genes encoding the extracellular matrix protein collagen (Col) VI. Opening of the cyclosporin A-sensitive mitochondrial permeability transition pore (PTP) is a causative event in disease pathogenesis, and a potential target for therapy. Here, we have tested the effect of N-methyl-4-isoleucine-cyclosporin (NIM811), a non-immunosuppressive cyclophilin inhibitor, in a zebrafish model of ColVI myopathy obtained by deletion of the N-terminal region of the ColVI α1 triple helical domain, a common mutation of UCMD. Treatment with antisense morpholino sequences targeting col6a1 exon 9 at the 1-4 cell stage (within 1 h post fertilization, hpf) caused severe ultrastructural and motor abnormalities as assessed by electron and fluorescence microscopy, birefringence, spontaneous coiling events and touch-evoked responses measured at 24-48 hpf. Structural and functional abnormalities were largely prevented when NIM811--which proved significantly more effective than cyclosporin A--was administered at 21 hpf, while FK506 was ineffective. Beneficial effects of NIM811 were also detected (i) in primary muscle-derived cell cultures from UCMD and BM patients, where the typical mitochondrial alterations and depolarizing response to rotenone and oligomycin were significantly reduced; and (ii) in the Col6a1(-/-) myopathic mouse model, where apoptosis was prevented and muscle strength was increased. Since the PTP of zebrafish shares its key regulatory features with the mammalian pore, our results suggest that early treatment with NIM811 should be tested as a potential therapy for UCMD and BM.

Sleep disorders in patients with spinal cord injury.

Sleep disturbances are globally more frequent in patients with spinal cord injury (SCI) than in the able-bodied population, and could contribute to dysfunction and poor quality of life in these patients. Specific sleep disorders may also contribute to negative health outcomes enhancing cardiovascular risk in a condition that per se increases heart disease related mortality. This review focuses on prevalence, features and treatment of sleep disorders in SCI. Although data on these subjects have been produced, reports on pathophysiology, consequences and treatment of sleep disorders are scarce or contradictory and more studies are required.

Ultrastructural changes in muscle cells of patients with collagen VI-related myopathies.

Collagen VI is an extracellular matrix protein expressed in several tissues including skeletal muscle. Mutations in COL6A genes cause Bethlem Myopathy (BM), Ullrich Congenital Muscular Dystrophy (UCMD) and Myosclerosis Myopathy (MM). Collagen VI deficiency causes increased opening of the mitochondrial permeability transition pore (mPTP), leading to ultrastructural and functional alterations of mitochondria, amplified by impairment of autophagy. Here we report for the first time ultrastructural studies on muscle biopsies from BM and UCMD patients, showing swollen mitochondria with hypodense matrix, disorganized cristae and paracrystalline inclusions, associated with dilated sarcoplasmic reticulum and apoptotic changes. These data were supported by scanning electron microscopy analysis on BM and UCMD cultured cells, showing alterations of the mitochondrial network. Morphometric analysis also revealed a reduced short axis and depicted swelling in about 3% of mitochondria. These data demonstrate that mitochondrial defects underlie the pathogenetic mechanism in muscle tissue of patients affected by collagen VI myopathies.

Expression of collagen VI α5 and α6 chains in human muscle and in Duchenne muscular dystrophy-related muscle fibrosis.

Collagen VI is a major extracellular matrix (ECM) protein with a critical role in maintaining skeletal muscle functional integrity. Mutations in COL6A1, COL6A2 and COL6A3 genes cause Ullrich Congenital Muscular Dystrophy (UCMD), Bethlem Myopathy, and Myosclerosis. Moreover, Col6a1(-/-) mice and collagen VI deficient zebrafish display a myopathic phenotype. Recently, two additional collagen VI chains were identified in humans, the α5 and α6 chains, however their distribution patterns and functions in human skeletal muscle have not been thoroughly investigated yet. By means of immunofluorescence analysis, the α6 chain was detected in the endomysium and perimysium, while the α5 chain labeling was restricted to the myotendinous junctions. In normal muscle cultures, the α6 chain was present in traces in the ECM, while the α5 chain was not detected. In the absence of ascorbic acid, the α6 chain was mainly accumulated into the cytoplasm of a sub-set of desmin negative cells, likely of interstitial origin, which can be considered myofibroblasts as they expressed α-smooth muscle actin. TGF-ß1 treatment, a pro-fibrotic factor which induces trans-differentiation of fibroblasts into myofibroblasts, increased the α6 chain deposition in the extracellular matrix after addition of ascorbic acid. In order to define the involvement of the α6 chain in muscle fibrosis we studied biopsies of patients affected by Duchenne Muscular Dystrophy (DMD). We found that the α6 chain was dramatically up-regulated in fibrotic areas where, in contrast, the α5 chain was undetectable. Our results show a restricted and differential distribution of the novel α6 and α5 chains in skeletal muscle when compared to the widely distributed, homologous α3 chain, suggesting that these new chains may play specific roles in specialized ECM structures. While the α5 chain may have a specialized function in tissue areas subjected to tensile stress, the α6 chain appears implicated in ECM remodeling during muscle fibrosis.

Cyclosporine A in Ullrich congenital muscular dystrophy: long-term results.

Six individuals with Ullrich congenital muscular dystrophy (UCMD) and mutations in the genes-encoding collagen VI, aging 5-9, received 3-5 mg/kg of cyclosporine A (CsA) daily for 1 to 3.2 years. The primary outcome measure was the muscle strength evaluated with a myometer and expressed as megalimbs. The megalimbs score showed significant improvement (P = 0.01) in 5 of the 6 patients. Motor function did not change. Respiratory function deteriorated in all. CsA treatment corrected mitochondrial dysfunction, increased muscle regeneration, and decreased the number of apoptotic nuclei. Results from this study demonstrate that long-term treatment with CsA ameliorates performance in the limbs, but not in the respiratory muscles of UCMD patients, and that it is well tolerated. These results suggest considering a trial of CsA or nonimmunosuppressive cyclosporins, that retains the PTP-desensitizing properties of CsA, as early as possible in UCMD patients when diaphragm is less compromised.

eEF1A phosphorylation in the nucleus of insulin-stimulated C2C12 myoblasts: Ser5³ is a novel substrate for protein kinase C ßI.

Recent data indicate that some PKC isoforms are translocated to the nucleus, in response to certain stimuli, where they play an important role in nuclear signaling events. To identify novel interacting proteins of conventional PKC (cPKC) at the nuclear level during myogenesis and to find new PKC isozyme-specific phosphosubstrates, we performed a proteomics analysis of immunoprecipitated nuclear samples from mouse myoblast C2C12 cells following insulin administration. Using a phospho(Ser)-PKC substrate antibody, specific interacting proteins were identified by LC-MS/MS spectrometry. A total of 16 proteins with the exact and complete motif recognized by the phospho-cPKC substrate antibody were identified; among these, particular interest was given to eukaryotic elongation factor 1α (eEF1A). Nuclear eEF1A was focalized in the nucleoli, and its expression was observed to increase following insulin treatment. Of the cPKC isoforms, only PKCßI was demonstrated to be expressed in the nucleus of C2C12 myocytes and to co-immunoprecipitate with eEF1A. In-depth analysis using site-directed mutagenesis revealed that PKCßI could phosphorylate Ser5³ of the eEF1A2 isoform and that the association between eEF1A2 and PKCßI was dependent on the phosphorylation status of eEF1A2.

Emerin-prelamin A interplay in human fibroblasts.

BACKGROUND INFORMATION: Emerin is a nuclear envelope protein that contributes to nuclear architecture, chromatin structure, and gene expression through its interaction with various nuclear proteins. In particular, emerin is molecularly connected with the nuclear lamina, a protein meshwork composed of lamins and lamin-binding proteins underlying the inner nuclear membrane. Among nuclear lamina components, lamin A is a major emerin partner. Lamin A, encoded by the LMNA gene (lamin A/C gene), is produced as a precursor protein (prelamin A) that is post-transcriptionally modified at its C-terminal region where the CaaX motif triggers a sequence of modifications, including farnesylation, carboxymethylation, and proteolytic cleavage by ZMPSTE 24 (zinc metalloproteinase Ste24) metalloproteinase. Impairment of the lamin A maturation pathway causing lamin A precursor accumulation is linked to the development of rare diseases such as familial partial lipodystrophy, MADA (mandibuloacral dysplasia), the Werner syndrome, Hutchinson-Gilford progeria syndrome and RD (restrictive dermopathy). RESULTS: In the present study, we show that emerin and different prelamin A forms influence each other's localization. We show that the accumulation of non-farnesylated as well as farnesylated carboxymethylated lamin A precursors in human fibroblasts modifies emerin localization. On the contrary, emerin absence at the inner nuclear membrane leads to unprocessed (non-farnesylated) prelamin A aberrant localization only. Moreover, we observe that the restoration of emerin expression in emerin-null cells induces the recovery of non-farnesylated prelamin A localization. CONCLUSION: These results indicate that emerin-prelamin A interplay influences nuclear organization. This finding may be relevant to the understanding of laminopathies.

Effects of prelamin A processing inhibitors on the differentiation and activity of human osteoclasts.

Osteoclast differentiation is a complex process involving cytoskeleton and nuclear reorganization. Osteoclasts regulate bone homeostasis and have a key role in bone degenerative processes. Osteolysis and osteoporosis characterize a subset of laminopathies, inherited disorders due to defects in lamin A/C. Laminopathies featuring bone resorption are characterized, at the molecular level, by anomalous accumulation of the unprocessed lamin A precursor, called prelamin A. To obtain a suitable cell model to study prelamin A effects on osteoclasts, prelamin A processing inhibitors FTI-277 or AFCMe were applied to peripheral blood monocytes induced to differentiate towards the osteoclastic lineage. Previous studies have shown that treatment with FTI-277 causes accumulation of non-farnesylated prelamin A, while AFCMe inhibition of prelamin A maturation causes accumulation of a farnesylated form. We demonstrate that monocytes subjected to FTI-277 treatment and mostly those subjected to AFCMe administration, differentiate towards the osteoclastic lineage more efficiently than untreated monocytes, in terms of number of multinucleated giant cells, mRNA expression of osteoclast-related genes and TRACP 5b activity. On the other hand, the bone resorption activity of osteoclasts obtained in the presence of high prelamin A levels is lower with respect to control osteoclasts. This finding may help the understanding of the osteolytic and osteoporotic processes that characterize progeroid laminopathies.

PKC-zeta expression is lower in osteoblasts from arthritic patients: IL1-beta and TNF-alpha induce a similar decrease in non-arthritic human osteoblasts.

Protein kinase C (PKC) is a family of enzymes detected in a diverse range of cell types where they regulate various cellular functions such as proliferation, differentiation, cytoskeletal remodelling, cytokine production, and receptor-mediated signal transduction. In this study we have analyzed the expression of 11 PKC isoforms (-alpha, -beta(I), -beta(II), -gamma, -delta, -eta, -theta, -epsilon, -zeta, -iota/lambda, and -micro) in osteoblasts from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) in comparison with osteoblasts from post-traumatic (PT) patients. By Western blotting analysis, nine isoforms, -alpha, -beta(I), -beta(II), -delta, -theta, - epsilon, -zeta, - iota/lambda, and -micro, were detected in osteoblasts. In RA and OA patients, PKC -theta and -micro were greater expressed whereas PKC-epsilon and -zeta decreased when compared with normal cells. The subcellular distribution and quantitative differences were confirmed by immuno-electron microscopy. Furthermore, we demonstrated that treatment with the proinflammatory cytokines, IL-1beta and TNF-alpha, significantly decreased PKC-zeta expression in PT osteoblasts. This suggests that proinflammatory cytokines can modulate the expression of this PKC isoform in osteoblasts in a way which is similar to changes detected in arthritic patients.

Compound heterozygosity for mutations in LMNA in a patient with a myopathic and lipodystrophic mandibuloacral dysplasia type A phenotype.

CONTEXT: Mandibuloacral dysplasia type A (MADA; OMIM 248370) is a rare progeroid syndrome characterized by dysmorphic craniofacial and skeletal features, lipodystrophy, and metabolic complications. Most Italian patients carry the same homozygous missense mutation (p.R527H) in the C-terminal tail domain of the LMNA gene, which encodes lamin A/C, an intermediate filament component of the nuclear envelope. OBJECTIVE: The objective of the study was to identify novel LMNA mutations in individuals with clinical characteristics (bird-like facies, mandibular and clavicular hypoplasia, acroosteolysis, lipodystrophy, alopecia) observed in other well-known patients. DESIGN: The LMNA gene was sequenced. Functional properties of the mutant alleles were investigated. PATIENT: We report a 27-yr-old Italian woman showing a MADA-like phenotype. Features include a hypoplastic mandible, acroosteolysis, pointed nose, partial loss of sc fat, and a progeric appearance. Due to the absence of clavicular dysplasia and normal metabolic profiles, generally associated with muscle hyposthenia and generalized hypotonia, this phenotype can be considered an atypical laminopathy. RESULTS: We identified a patient compound heterozygote for the p.R527H and p.V440M alleles. The patient's cells showed nuclear shape abnormalities, accumulation of pre-lamin A, and irregular lamina thickness. Lamins A and C showed normal expression and localization. The electron microscopy detected heterochromatin defects with a pattern similar to those observed in other laminopathies. However, chromatin analysis showed a normal distribution pattern of the major heterochromatin proteins: heterochromatin protein-1beta and histone H3 methylated at lysine 9. CONCLUSIONS: The clinical and cellular features of this patient show overlapping laminopathy phenotypes that could be due to the combination of p.R527H and p.V440M alleles.

Effects of antisense mediated inhibition of cathepsin K on human osteoclasts obtained from peripheral blood.

Cathepsin K is a cystein protease that displays a proteolytic activity against Type I collagen and is abundantly and selectively expressed in osteoclasts where it plays a critical role in bone degradation. Its direct role in bone tissue has been defined by knock-out mice studies and inhibiting strategies in animals models. However, direct proof of cathepsin K function in human osteoclast model in vitro is lacking. The aim of this study is to analyze cathepsin K expression and localization in human osteoclasts obtained from peripheral blood and to examine cathepsin K function in these cells by antisense oligodeoxynucleotide (AS-ODN) strategy. AS-ODN was added to the culture of osteoclast precursors induced to differentiate by RANKL and M-CSF. AS-ODN treatment produced a significant down-regulation of cathepsin K mRNA (>80%) and protein expression, as verified respectively by Real-time PCR and by immunocytochemistry or Western blot. The cathepsin K inhibition caused an impairment of resorption activity as evaluated by a pit formation assay ( p = 0.045) and by electron microscopy, while the acidification process was unaffected. We demonstrated that antisense strategies against cathepsin K are selectively effective to inhibit resorption activity in human osteoclasts, like in animal models.

Ultrastructural defects of collagen VI filaments in an Ullrich syndrome patient with loss of the alpha3(VI) N10-N7 domains.

Ultrastructural alterations of collagen VI in cultured fibroblasts and reduced collagen VI immunostaining in the papillary dermis and endomysium were detected in a patient with a mild form of Ullrich congenital muscular dystrophy caused by a COL6A3 gene mutation. The patient had been previously demonstrated to express an alpha3(VI) chain shorter than normal due to skipping of the mutated exon. We show that collagen VI filaments are not organized in a normal network in the extracellular matrix secreted by patient's cultured fibroblasts. Moreover, we demonstrate that in this patient the alpha3(VI) chain is produced in lower amounts and it is almost exclusively represented by the shorter, alternatively spliced N6-C5 isoform. These results suggest that different alpha3(VI) chain isoforms, containing also domains of the N10-N7 region, are required for assembling a proper collagen VI network in the extracellular matrix.

Quantitative immunodetection of key elements of polyphosphoinositide signal transduction in osteoblasts from arthritic patients shows a direct correlation with cell proliferation.

Phosphoinositides play an essential role in diverse cellular functions such as cell proliferation, cytoskeletal regulation, intracellular vesicle trafficking, motility, cell metabolism and death. Alteration of these pathways is common to many diseases. In this study, we show that osteoblasts from patients affected by osteoarthritis (OA) and by rheumatoid arthritis (RA) present a decreased cell proliferation and a reduced expression of the key elements of polyphosphoinositide signal transduction such as phosphatidylinositol-3-kinase (PI 3K), phospholipase C gamma1 (PLCgamma1), and protein kinase C zeta (PKCzeta) compared to the post-traumatic (PT) patients. Our results suggest that a correlation may exist between the reduced osteoblast proliferation observed in OA and RA patients and the lowered expression of PI 3K, PLCgamma1, and PKCzeta enzymes. The reduced proliferation rate of osteoblasts in response to these signal transduction effectors could counteract the evolution of arthritic disease.

Leiomyosarcoma of the spermatic cord: a light and ultrastructural description of one case.

A case of leiomyosarcoma arising in the spermatic cord is described. A 83-year-old man required medical care for an irreducible inguinal hernia. The patient underwent herniorraphy and transinguinal radical orchiectomy. Macroscopically, the spermatic cord was enlarged by a gray-tan and ill-defined neoplasm measuring 4 x 4 x 3 cm. Histologically, this proliferation was composed of atypical spindle cells with blunted end nuclei and eosinophilic cytoplasm. Immunohistochemistry and ultrastructure confirmed the smooth muscle nature of the neoplastic cells. The diagnosis of leiomyosarcoma of the spermatic cord was made. To improve the assignment of this rare lesion to its specific anatomic location, we analyzed the immunohistochemical and ultrastructural characteristics of the smooth muscle tumoral cells and in particular those of the intracellular filament aggregates.

Involvement of nuclear phosphatidylinositol-dependent phospholipases C in cell cycle progression during rat liver regeneration.

Nuclear lipid metabolism is involved in the regulation of cell proliferation. Modulation of the expression and activity of nuclear PI-phospholipase C (PI-PLC) has been reported during liver regeneration after partial hepatectomy, although it has not been determined whether different PLC isoforms play specific roles in the regulation of cell cycle progression. Here, we report evidence that the increased activity of nuclear PLCs in regenerating rat liver occurs before the peak of DNA replication and involves the enzyme activity associated to the chromatin and not that associated to the nuclear membrane. Immunocytochemical analyses indicate that PI-PLC beta(1) isoform is exclusively localized at the chromatin level, PI-PLC beta(1) co-localizes with DNA replication sites much more than PI-PLC gamma(1), which is also present at the nuclear envelope. These findings and the increased amount of PI-PLC gamma(1) occurring after the peak of DNA replication suggest that PI-PLC beta(1) and gamma(1) play different roles in cell cycle progression during regenerating liver. The increased activity of PI-PLC beta(1) constitutively present within the hepatocyte nucleus, should trigger DNA replication, whereas PI-PLC gamma(1) should be involved in G2/M phase transition through lamin phosphorylation.