Abstract
The apoptosis of chondrocytes plays an important role in endochondral bone formation and in cartilage degradation during aging and disease. Prolactin (PRL) is produced in chondrocytes and is known to promote the survival of various cell types. Here we show that articular chondrocytes from rat postpubescent and adult cartilage express the long form of the PRL receptor as revealed by immunohistochemistry of cartilage sections and by RT-PCR and Western blot analyses of the isolated chondrocytes. Furthermore, we demonstrate that PRL inhibits the apoptosis of these same chondrocytes cultured in low-serum. Chondrocyte apoptosis was measured by hypodiploid DNA content determined by flow cytometry and by DNA fragmentation evaluated by the ELISA and the TUNEL methods. The anti-apoptotic effect of PRL was dose-dependent and was prevented by heat inactivation. These data demonstrate that PRL can act as a survival factor for chondrocytes and that it has potential preventive and therapeutic value in arthropathies characterized by cartilage degradation.
Introduction
Chondrocytes are the only cells residing in cartilage and are responsible for the formation, maintenance, and turnover of a variety of extracellular matrix proteins. Indeed, chondrocytes are essential for the function of the tissue; their death is an important feature of cartilage replacement during bone formation (Poole 1991), but in aging and disease it can lead to cartilage degradation (Aigner & McKenna 2002). Various lines of evidence suggest that apoptosis is the main type of death in chondrocytes. Apoptotic chondrocytes have been detected during endochondral ossification (Hatori et al. 1995, Zenmyo et al. 1996), in aging cartilage (Adams & Horton 1998), and in arthropathies characterized by cartilage destruction, such as rheumatoid arthritis (Kim & Song 1999) and osteoarthritis (Kouri et al. 1997, Goggs et al. 2003). In osteoarthritis, the major age-associated joint disease, increased chondrocyte apoptosis has been correlated with the severity of cartilage damage (Hashimoto et al. 1998). Morphological and functional differences suggest that the mechanisms of chondrocyte apoptosis differ from those in other tissues (Roach et al. 2004, Pérez et al. 2005), but these mechanisms are unclear and little is known about the regulatory factors responsible for their control.
Prolactin (PRL) acts both as a circulating hormone and as a cytokine in a vast array of physiological functions that range from reproduction and osmoregulation to immunomodulation and angiogenesis (Bole-Feysot et al. 1998, Corbacho et al. 2002). In addition, PRL may serve as a regulatory factor for joint tissues. PRL can act directly on osteoblasts during development to regulate bone formation (Clément-Lacroix et al. 1999, Coss et al. 2000) and can activate synovial cell functions in rheumatoid arthritis (Nagafuchi et al. 1999). Furthermore, synovial fluid contains PRL (Ogueta et al. 2002) that may derive from plasma and/or may be produced locally. Indeed, PRL is expressed by synovial cells (Nagafuchi et al. 1999), chondrocytes (Macotela et al. 2006), and bone marrow-derived mesenchymal stem cells undergoing chondrogenic differentiation (Ogueta et al. 2002). In the latter, PRL may contribute to the acquisition of the chondrocytic phenotype as it stimulates the synthesis of proteoglycans and type II collagen (Ogueta et al. 2002). In support of chondrocytes being cellular targets of PRL, the PRL receptor has been detected in chondrocytes from neonatal rats (Coss et al. 2000), although its functional role remains to be determined. Because PRL is known to be a survival factor for various cell types (Ploszaj et al. 1998, Buckley 2001, Tessier et al. 2001, Ruffion et al. 2003, Perks et al. 2004, Asai-Sato et al. 2005) and chondrocyte survival is essential for cartilage function, the aim of the present work was to investigate whether the PRL receptor is expressed in articular chondrocytes from normal post-pubescent and adult cartilage and can mediate an action of PRL on cell survival.
Materials and Methods
Chondrocyte isolation
Articular chondrocytes were isolated from femoral epiphyseal cartilage of male, postpubescent, Wistar rats (8 weeks old, 130–150 g body weight (bw)) as described (Shakibaei et al. 1997) with modifications (Macotela et al. 2006). The cells were either stored immediately at −80 ° C for subsequent total RNA extraction, or processed for immunoprecipitation/Western blot analysis or cell culture experiments.
RT-PCR
Total RNA was extracted and quantified, and 5 μg were reverse transcribed in a 25-μl reaction using Moloney murine leukemia virus reverse transcriptase (Promega). Two-microliter aliquots were used for cDNA amplification by PCR using oligonucleotides specific for all forms of the PRL receptor (sense primer A: CCC CAA CTC CTG CTT CTT TAG, and antisense primer B: TAT TTT TGG CCC AGG AAC TA), or oligonucleotides specific for the short form (sense primer C: ATC CTG GGA CAG ATG GAG GAC, and antisense primer D: TGG CTG AGG CTG ACA AAA GAG), or for the long PRL receptor isoform (sense primer C, and antisense primer E: AGA CAG TGG GGC TTT TCT CCT). In all cases, amplification was for 40 cycles and for 30 s at 94 ° C, 45 s at 56 ° C, and 45 s at 72 ° C for primers A–B; and 30 s at 94 ° C, 45 s at 56 ° C, and 60 s at 72 ° C for primers C–D and C–E. The products resulting from reactions with primers A–B, C–D, and C–E were 582 bp, 1017 bp and 1344 bp respectively (Fig. 1A).
Immunoprecipitation/Western blot
Freshly isolated chondrocytes (2 × 106) were resuspended in lysis buffer (0.5% Nonidet P-40, 0.1% SDS, 50 mM Tris, 150 mM NaCl, 1 μg/ml aprotinin, and 100 μg/ml phenylmethylsulfonyl fluoride, pH 7) and immunoprecipitated with anti-rat PRL receptor U-5 monoclonal antibody (U-5 MAb, 5 μg) using the previously reported technique (Corbacho et al. 2000). U-5 MAb is directed against the extracellular region of the PRL receptor (Okamura et al. 1989) and was a gift from P A Kelly from INSERM U-584, Paris, France. Immuno-precipitates were subjected to SDS-PAGE on an 8% acrylamide gel under reducing conditions, then blotted and probed with a 1:500 dilution of U-5 MAb. The antigen–antibody complex was detected using the alkaline phosphatase second antibody kit (Bio-Rad Laboratories).
Immunohistochemistry
Bones from the knee joint of adult rats (250 g bw) were dissected, fixed, decalcified, and dehydrated for paraffin embedding. After deparaffination and rehydration, longitudinal 6-μm paraffin sections were blocked with 2% bovine serum albumin, 1% normal goat serum, and 0.3% Triton-X in phosphate-buffered saline (pH 7.4) for 1 h. Sections were then incubated overnight with a 1:100 dilution of U-5 or U-6 anti-rat PRL receptor MAbs. The U-6 MAb is also directed against the extracellular region of the PRL receptor (Okamura et al. 1989) and was provided by P A Kelly. Finally, after incubation with biotin-conjugated secondary antibody for 1 h, sections were developed using the avidin–biotin complex detection kit (Vector Laboratories, Burlingame, CA, USA).
Chondrocyte culture
Chondrocytes were seeded (2 × 105 cells/cm2) on wells or on 1% fibronectin-coated glass coverslips and incubated in Dulbecco’s modified Eagle’s medium (DMEM) containing 0.5% fetal bovine serum (FBS) and 1% penicillin/streptomycin for 48 h at 37 ° C in the presence or absence of rat PRL (biological grade; National Hormone Pituitary Program, Torrance, CA, USA). Cells cultured in 10% FBS-DMEM served as negative control for apoptosis.
DNA labeling technique for flow cytometric analysis
At the end of the incubation, chondrocytes were trypsinized and sedimented. Cell pellets were fixed in 80% ethanol for 60 min at 4 ° C, washed twice, incubated with RNAse (300 μg/ml) for 20 min at 4 ° C and stained with propidium iodide (50 μg/ml) for 15 min at 4 ° C in the dark. Propidium iodide fluorescence of nuclei was measured by flow cytometry on a fluorescence-activated cell sorter (Becton Dickinson, Franklin Lakes, NJ, USA) with a 560 nm dichromatic mirror and a 600 nm band pass filter. For each sample, 104 cells were analyzed and percentage values denoted the proportion of propidium iodide positive cells.
TUNEL assay
Chondrocytes were fixed with fresh 4% paraformaldehyde for 1 h at room temperature and permeabilized by treatment with 0.1% Triton X-100 in 0.1% sodium citrate buffer (pH 7.4) for 2 min at 4 ° C. Following permeabilization, apoptotic cells were visualized by the TUNEL method using a detection kit (Roche Diagnostics) and fluorescence microscopy.
Cell death ELISA
Fragmented nucleosomal DNA was measured using the ELISA kit from Boehringer Mannheim, according to the manufacturer’s instructions. After incubation, medium containing floating cells was harvested, and the cells on the plate were trypsinized briefly. Floating and trypsinized cells were combined, sedimented, counted, lysed in the lysis buffer of the kit and transferred to a microtiter plate to quantitate nucleosomes. The results are expressed as optical density units per 5 × 104 cells relative to those of control cells (incubated with 10% FBS).
Statistical analyses
All results were replicated in three or more independent experiments. Data are presented as the mean ± s.e.m. As appropriate, Student’s unpaired t-test or one-way ANOVA followed by Tukey’s test to compare individual means was used for statistical comparisons. The significance level was set at 5%.
Results
The long form of the PRL receptor is expressed in chondrocytes
In order to study whether PRL can act directly on chondrocytes to regulate their survival, we first examined the expression of PRL receptors in articular chondrocytes isolated from normal, postpubescent rat cartilage. The PRL receptor mRNA was investigated by RT-PCR (Fig. 1). Amplification using primers common to all forms of the PRL receptor or specific for the long form of the PRL receptor yielded products having the expected lengths of 582 bp or 1344 bp respectively (Fig. 1B, lanes 4 and 5). These products were similar to the control PCR bands amplified from the rat PRL receptor cDNA (lanes 1 and 2). No product was amplified from chondrocytes when primers specific for the short form of the PRL receptor were used (lane 6). To determine whether the PRL receptor mRNA was translated into protein, PRL receptors were immunoprecipitated from chondrocyte lysates and analyzed by immunoblot (Fig. 1C). The anti-rat PRL receptor U-5 MAb precipitated a major protein of 85 kDa, the expected size for the long form of the PRL receptor (Okamura et al. 1989), and several minor proteins above and below this size. Some of the latter are close to the size estimated for the intermediate (65 kDa) and the short (40 kDa) PRL receptor variants, but additional work is needed to clarify their nature, inasmuch as no short PRL receptor mRNA was detected. In addition, the presence of PRL receptors was examined by immunohistochemistry in sections of femoral heads from adult rats (Fig. 2). Chondrocytes throughout femoral articular cartilage stained positively for PRL receptors as revealed by the anti-rat PRL receptor U-5 and U-6 MAbs (Fig. 2B, D), and no positive labeling was evident in their absence (Fig. 2A) nor with control IgGs of the same isotype (Fig. 2C).
PRL inhibits apoptosis of articular chondrocytes induced by serum deprivation
Next, we determined whether primary chondrocytes isolated from rat postpubescent articular cartilage could be induced to undergo apoptosis by the classic signal of serum deprivation. Rat articular chondrocytes were incubated with 0.5% serum, and the amount of apoptosis was measured by propidium iodide staining and fluorescence-activated cell sorting analysis (Fig. 3). This method of flow cytometry quantitates the percentage of cells with hypodiploid DNA occurring because of DNA fragmentation. Incubation in 0.5% serum resulted in a peak of apoptotic cells with DNA hypoploidy that was absent when cells were cultured in 10% serum. The number of apoptotic cells induced by low-serum corresponded to approximately 35% of total chondrocytes. Coincubation with 10 or 100 nM PRL prevented the appearance of hypodiploid cells induced by low-serum. The two PRL concentrations were equally effective, and their protective action was abolished by heat denaturation (Fig. 3B).
PRL protection against apoptosis was confirmed by the detection of DNA strand breaks in cells using the TUNEL method (Fig. 4). Incubation with 0.5% serum increased the number of TUNEL-positive chondrocytes by sixfold compared with cells cultured in 10% serum. The increase in TUNEL-positive chondrocytes was prevented by PRL in a dose-dependent manner (Fig. 4B).
Finally, chondrocyte apoptosis was determined using an ELISA that measures DNA nucleosomal fragments (Fig. 5). Low-serum resulted in a fourfold increase in the amount of apoptosis, and this increase was inhibited by increasing concentrations of PRL.
Discussion
Our present study is the first to report that PRL can protect chondrocytes against apoptotic death. Apoptosis was effectively antagonized by PRL. The cellular response to low-serum is compatible with apoptosis, as characterized by flow cytometry quantification of cells with hypo-diploid DNA, TUNEL staining, and ELISA measurement of DNA fragments. These three methods identify the degradation of DNA by cleavage between nucleosomes, a hallmark of apoptosis. Consistent with the protective effect of PRL, we also show that chondrocytes express the long form of the PRL receptor mRNA and that this message is translated into significant amounts of protein in the isolated cells and in chondrocytes in situ.
The anti-apoptotic effect of PRL was dose related in the TUNEL and ELISA assays. In the flow cytometry assay, the two PRL concentrations tested (10 and 100 nM) were equally potent. The reasons for the same potency are unclear, but might stem from errors in collecting the small amount of light associated with dead cells, which are difficult to spin down by ordinary low-speed centrifugations. The mechanisms underlying the anti-apoptotic effect of PRL in chondrocytes need to be investigated. The survival action of PRL has been associated with the upregulation of Bcl-2, a family of proteins in mouse mammary epithelial cells and in breast cancer cells (Ploszaj et al. 1998, Peirce & Chen 2004), and downregulation of Bcl-2 plays an important role in chondrocyte apoptosis induced by serum withdrawal (Feng et al. 1998).
The observation that articular chondrocytes from post-pubertal rats undergo apoptosis in response to low-serum is in agreement with other in vitro studies using 0% serum and chick embryo and adult rat sterna chondrocytes (Ishizaki et al. 1994) or adult human and rabbit articular chondrocytes (Feng et al. 1998). These findings indicate that, similar to most vertebrate cells, chondrocytes require growth factor signaling for survival. Various hormones and growth factors promote the survival of cultured chondrocytes, including dexamethasone, insulin, basic fibroblast growth factor, transforming growth factor (TGF)-β and insulin-like growth factor-I (Quarto et al. 1992, Ishizaki et al. 1994, Gruber et al. 2000, Lo & Kim 2004). The present results add PRL to this list and raise the important question of whether PRL is one of the factors regulating the survival of articular chondrocytes in vivo.
Articular cartilage is the thin layer of smooth hyaline cartilage that covers the joint surfaces of a bone. Adult articular cartilage is thought to be a postmitotic tissue and because there is virtually no cell turnover, preservation of cell viability is essential for its function (Aigner & Kim 2002). Because cartilage is avascular, serum-borne survival factors must be able to diffuse through the tissue from the synovial fluid of the joint. However, due to the sparse distribution of chondrocytes encased within the extra-cellular cartilage matrix, an autocrine survival mechanism may be more efficient, and chondrocytes do produce survival-promoting factors (Ishizaki et al. 1994). In this regard, PRL is a component of human synovial fluid (Ogueta et al. 2002, Rovensky et al. 2005), and may derive from plasma, since most proteins with a molecular mass of less than 100 kDa readily transfer from one fluid space to the other (Perman 1980). In addition, PRL can be generated by chondrocytes. Isolated chondrocytes from rat articular cartilage express PRL mRNA and protein (Macotela et al. 2006), and bone marrow-derived mesenchymal progenitor cells express PRL mRNA during chondrogenic differentiation (Ogueta et al. 2002).
Furthermore, chondrocytes express the PRL receptor. PRL receptors have been detected in chondrocytes from cartilage in the diaphysis of digits in newborn rats (Coss et al. 2000) and in bone marrow-derived mesenchymal stem cells undergoing chondrogenic differentiation (Ogueta et al. 2002). PRL receptors exist in several isoforms that differ primarily in the sequence and length of the cytoplasmic domain (Bole-Feysot et al. 1998). Of interest is the observation that upon chondrogenic differentiation, mesenchymal stem cells switch expression from the intermediate isoform to the long isoform of the PRL receptor (Ogueta et al. 2002). In agreement, we show that chondrocytes from postpubertal articular cartilage express the long form of the PRL receptor. Moreover, this receptor isoform is functional in chondrocytes, because PRL stimulates the synthesis of proteoglycans and type II collagen in the chondrocytic mesenchymal cells (Ogueta et al. 2002) and protects articular chondrocytes against low-serum-induced apoptosis (present results).
Although chondrocytes respond to PRL in vitro, the in vivo action of PRL on cartilage function remains to be established. Targeted disruption of the PRL receptor reduces bone development and growth, but this action appears to be independent of a defect in cartilage (Clément-Lacroix et al. 1999). Bone alterations were observed mostly in calvaria, where bone formation occurs by intramembranous ossification, and there was no skeletal alteration indicative of a defect of endochondral bone formation. Given that endochondral ossification requires chondrocyte apoptosis, the absence of an endochondral ossification phenotype in these mice would argue against a role for PRL in cartilage survival. While redundant mechanisms could compensate for the loss of PRL action, it is also possible that the anti-apoptotic effect of PRL does not occur in chondrocytes involved in bone development and growth. Articular chondrocytes and cells in prenatal and growth plate cartilage are known to have differences that reflect specific functions and long-term survival (Karsenty & Wagner 2002). For example, unlike other chondrocytes, articular chondrocytes are normally arrested before hypertropic differentiation leading to apoptosis, allowing cartilage on the articular surface to persist, which is essential for proper joint function. Multifunctional proteins can exhibit differential effects on chondrocyte subpopulations. For example, TGF-β stimulates chondrogenesis of undifferentiated multipotent mesenchymal cells (Leonard et al. 1991), but it blocks hypertropic differentiation of articular chondrocytes, promoting the survival of articular cartilage (Serra et al. 1997). Evaluation of the effect of PRL receptor gene deletion on the aging skeleton or in degenerative joint diseases may provide strong support for the role of PRL as a mediator of articular cartilage survival.
Importantly, loss of adult articular cartilage results from diverse actions including age, loading, and trauma, and it is the major cause of joint dysfunction and disability in rheumatoid arthritis and osteoarthritis (Goggs et al. 2003). Investigations leading to the identification of chondrocyte apoptosis inhibitors should have a major impact on the prevention and treatment of a wide range of disabling rheumatological conditions. Of interest is the fact that PRL has been detected in the synovial fluid of patients with rheumatoid arthritis and osteoarthritis (Rovensky et al. 2005), and that it is expressed by synovial cells in rheumatoid arthritis (Nagafuchi et al. 1999). A better understanding of the effect of PRL on chondrocyte survival both in vivo and in vitro should help elucidate its probable contribution to cartilage function under both healthy and diseased states.
(C Zermeño and J Guzmán-Morales contributed equally to this work)
We thank Daniel Mondragón, Antonio Prado, Blanca E Reyes Márquez, Pilar Galarza, and Martín García for their expert technical assistance and Dorothy D Pless for editing the manuscript. This work was supported by the National Council of Science and Technology of Mexico grant 43401 and by the National Autonomous University of Mexico grant IN202406. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
References
Adams CS & Horton WE 1998 Chondrocyte apoptosis increases with age in the articular cartilage of adult animals. Anatomical Record 250 418–425.
Aigner T & Kim HA 2002 Apoptosis and cellular vitality. Issues in osteoarthritic cartilage degeneration. Arthritis and Rheumatism 46 1986–1996.
Aigner T & McKenna L 2002 Molecular pathology and pathobiology of osteoarthritic cartilage. Cellular and Molecular Life Sciences 59 5–18.
Asai-Sato M, Nagashima Y, Miyagi E, Sato K, Ohta I & Vonderhaar BK 2005 Prolactin inhibits apoptosis of ovarian carcinoma cells induced by serum starvation or cisplatin treatment. International Journal of Cancer 115 539–544.
Bole-Feysot C, Goffin V, Edery M, Binart N & Kelly PA 1998 Prolactin (PRL) and its receptor: action, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. Endocrine Reviews 19 225–268.
Buckley AR 2001 Prolactin, a lymphocyte growth and survival factor. Lupus 10 684–690.
Clément-Lacroix P, Ormandy C, Lepescheux L, Ammann P, Damotte D, Goffin V, Bouchard B, Amling M, Gaillard-Kelly M, Binart N et al1999 Osteoblasts are a new target for prolactin: analysis of bone formation in prolactin receptor knockout mice. Endocrinology 140 96–105.
Corbacho AM, Macotela Y, Nava G, Torner L, Dueñas Z, Noris G, Morales MA, Martínez de la Escalera G & Clapp C 2000 Human umbilical vein endothelial cells express multiple prolactin isoforms. Journal of Endocrinology 166 53–62.
Corbacho AM, Martínez de la Escalera G & Clapp C 2002 Roles of prolactin and members of the prolactin/growth hormone/placental lactogen family in angiogenesis. Journal of Endocrinology 173 219–238.
Coss D, Yang L, Kuo CB, Xu X, Luben RA & Walker AM 2000 Effects of prolactin on osteoblast alkaline phosphatase and bone formation in the developing rat. American Journal of Physiology. Endocrinology and Metabolism 279 E1216–E1225.
Feng L, Precht P, Balakir R & Horton WE Jr 1998 Evidence of a direct role for Bcl-2 in the regulation of articular chondrocyte apoptosis under the conditions of serum withdrawal and retinoic acid treatment. Journal of Cellular Biochemistry 71 302–309.
Goggs R, Carter SD, Schulze-Tanzil G, Shakibaei M & Mobasheri A 2003 Apoptosis and the loss of chondrocyte survival signals contribute to articular cartilage degradation in osteoarthritis. Veterinary Journal 166 140–158.
Gruber HE, Norton HJ & Hanley EN Jr 2000 Anti-apoptotic effects of IGF-I and PDGF on human intervertebral disc cells in vitro. Spine 25 2153–2157.
Hashimoto S, Ochs RL, Komiya S & Lotz M 1998 Linkage of chondrocyte apoptosis and cartilage degradation in human osteoarthritis. Arthritis and Rheumatism 41 1632–1638.
Hatori M, Klatte KJ, Teixeira CC & Shapiro IM 1995 End labeling studies of fragmented DNA in the avian growth plate: evidence of apoptosis in terminally differentiated chondrocytes. Journal of Bone and Mineral Research 10 1960–1968.
Ishizaki Y, Burne JF & Raff MC 1994 Autocrine signals enable chondrocytes to survive in culture. Journal of Cell Biology 126 1069–1077.
Karsenty G & Wagner EF 2002 Reaching a genetic and molecular understanding of skeletal development. Developmental Cell 2 389–406.
Kim HA & Song YW 1999 Apoptotic chondrocyte death in rheumatoid arthritis. Arthritis and Rheumatism 42 1528–1537.
Kouri JB, Rosales-Encina JL, Chaudhuri PP, Luna J & Mena R 1997 Apoptosis in human osteoarthritic cartilage: a microscopy report. Journal of Medical Science Research 25 245–248.
Leonard CM, Fuld HM, Frenz DA, Downie SA, Massague J & Newman SA 1991 Role of transforming growth factor-β in chondrogenic pattern formation in the embryonic limb: stimulation of mesenchymal condensation and fibronectin gene expression by exogenous TGF-β and evidence of endogenous TGF-β-like activity. Developmental Biology 145 99–109.
Lo MY & Kim HT 2004 Chondrocyte apoptosis induced by collagen degradation: inhibition by caspase inhibitors and IGF-I. Journal of Orthopaedic Research 22 140–144.
Macotela Y, Aguilar M, Guzmán-Morales J, Rivera JC, Zermeño C, Lopez-Barrera F, Nava G, Lavalle C, Martínez de la Escalera G & Clapp C 2006 Matrix metalloproteases from chondrocytes generate antiangiogenic 16 k prolactin. Journal of Cell Science 119.
Nagafuchi H, Suzuki N, Kaneko A, Asai T & Sakane T 1999 Prolactin locally produced by synovium infiltrating T lymphocytes induces excessive synovial cell functions in patients with rheumatoid arthritis. Journal of Rheumatology 26 1890–1900.
Ogueta S, Muñoz J, Obregon E, Delgado-Baeza E & Garcia-Ruiz JP 2002 Prolactin is a component of the human synovial liquid and modulates the growth and chondrogenic differentiation of bone marrow-derived mesenchymal stem cells. Molecular and Cellular Endocrinology 190 51–63.
Okamura H, Zachwieja J, Raguet S & Kelly PA 1989 Characterization and applications of monoclonal antibodies to the prolactin receptor. Endocrinology 124 2499–2508.
Peirce SK & Chen WY 2004 Human PRL and its antagonist, hPRL-C129R, regulate bax and bcl-2 gene expression in human breast cancer cells and transgenic mice. Oncogene 23 1248–1255.
Pérez E, Luna MJ, Rojas L & Kouri JB 2005 Chondroptosis: an immunohistochemical study of apoptosis and Golgi complex in chondrocytes from human osteoarthritic cartilage. Apoptosis 10 1105–1110.
Perks CM, Keith AJ, Goodhew KL, Savage PB, Winters ZE & Holly JM 2004 Prolactin acts as a potent survival factor for human breast cancer cell lines. British Journal of Cancer 91 305–311.
Perman V 1980 Synovial fluid. In Clinical Biochemistry of Domestic Animals, edn 3. Ed J Kaneko. New York, NY, USA: Academic Press Inc.
Ploszaj T, Motyl T, Orzechowski A, Zimowska W, Wareski P, Skierski J & Zwierzchowski L 1998 Antiapoptotic action of prolactin is associated with up-regulation of Bcl-2 and down-regulation of Bax in HC11 mouse mammary epithelial cells. Apoptosis 3 295–304.
Poole AR 1991 The growth plate: cellular physiology, cartilage assembly and mineralization. In Cartilage: Molecular Aspects, pp 179–211. Eds BK Hall & SA Newman. Florida, USA: CRC Press.
Quarto R, Campanile G, Cancedda R & Dozin B 1992 Thyroid hormone, insulin, and glucocorticoids are sufficient to support chondrocyte differentiation to hypertrophy: a serum-free analysis. Journal of Cell Biology 119 989–995.
Roach HI, Aigner T & Kouri JB 2004 Chondroptosis: a variant of apoptotic cell death in chondrocytes. Apoptosis 9 265–277.
Rovensky J, Imrich R, Radikova Z, Simorova E, Greguska O, Vigas M & Macho L 2005 Peptide hormones and histamine in plasma and synovial fluid of patients with rheumatoid arthritis and osteoarthritis. Endocrine Regulations 39 1–6.
Ruffion A, Al-Sakkaf KA, Brown BL, Eaton CL, Hamdy FC & Dobson PR 2003 The survival effect of prolactin on PC3 prostate cancer cells. European Urology 43 301–308.
Serra R, Johnson M, Filvaroff EH, LaBorde J, Sheehan DM, Derynck R & Moses HL 1997 Expression of a truncated, kinase-defective TGF-β type II receptor in mouse skeletal tissue promotes terminal chondrocyte differentiation and osteoarthritis. Journal of Cell Biology 139 541–552.
Shakibaei M, DeSouza P & Merker HJ 1997 Integrin expression and collagen type II implicated in maintenance of chondrocyte shape in monolayer culture: an immunomorphological study. Cell Biology International 21 115–125.
Tessier C, Prigent-Tessier A, Ferguson-Gottschall S, Gu Y & Gibori G 2001 PRL anti-apoptotic effect in the rat decidua involves the PI3K/protein kinase B-mediated inhibition of caspase-3 activity. Endocrinology 142 4086–4094.
Zenmyo M, Komiya S, Kawabata R, Sasaguri Y, Inoue A & Morimatasu M 1996 Morphological and biochemical evidence for apoptosis in the terminal hypertrophic chondrocytes of the growth plate. Journal of Pathology 180 430–433.